Integrated Molecular and Bioinformatics Approaches for Disease-Related Genes in Plants

Modern plant pathology relies on bioinformatics approaches to create novel plant disease diagnostic tools. In recent years, a significant amount of biological data has been generated due to rapid developments in genomics and molecular biology techniques. The progress in the sequencing of agriculturally important crops has made it possible to develop a better understanding of plant–pathogen interactions and plant resistance. The availability of host–pathogen genome data offers effective assistance in retrieving, annotating, analyzing, and identifying the functional aspects for characterization at the gene and genome levels. Physical mapping facilitates the identification and isolation of several candidate resistance (R) genes from diverse plant species. A large number of genetic variations, such as disease-causing mutations in the genome, have been identified and characterized using bioinformatics tools, and these desirable mutations were exploited to develop disease resistance. Moreover, crop genome editing tools, namely the CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas9 (CRISPR-associated) system, offer novel and efficient strategies for developing durable resistance. This review paper describes some aspects concerning the databases, tools, and techniques used to characterize resistance (R) genes for plant disease management.


Background
Phytopathogens have greatly threatened livelihoods and societal growth because they affect quality crop production. Plant diseases caused by pathogenic bacteria, fungi, and viruses account for nearly 20-40% of losses in agricultural crop yields worldwide [1]. The molecular basis of the host-pathogen interaction is better understood due to the advancements in molecular and bioinformatics technologies. Whole-genome sequencing technology facilitates the sequencing of a large number of pathogens and plant species.
Scientists are now able to organize and analyze enormous amounts of biological data using bioinformatics tools. Additionally, they can be used to identify and characterize disease-related genes and develop new diagnostic tools [2]. Plants have developed a multi-layered defense system against microbial diseases during evolution. The first level of protection is provided by the physical barriers imposed by the plant surface. The second layer is related to the detection of pathogen-associated molecular patterns (PAMP) that are anchored to the plasma membrane and activate the PAMP-triggered immunity (PTI) [3]. The third layer involves receptors encoded by resistance genes (R genes) that recognize the presence of pathogen-effector proteins and activate effector-triggered immunity (ETI) [4]. Plant disease resistance can be classified into two categories, namely qualitative resistance and quantitative resistance. Qualitative resistance is controlled by single resistance (R) genes, while the latter is controlled by multiple genes or quantitative trait loci (QTLs) [5].

Genome Databases of Plant Pathogens
Genome databases integrated with specific bioinformatics tools have been developed to study the associations between genetic diversity and disease (Table 1). They also provide information related to host-pathogen interactions. PhytoPath is a bioinformatics resource for genomic and phenotypic data of important plant pathogen species. The PhytoPath project utilizes the Ensembl genome portals to provide genomic information, including genome sequences, structural and functional annotation of protein-coding and non-protein coding genes, DNA and protein-based alignments, and phylogeny for genes [30]. The National Institute of Agrobiological Sciences (NIAS) Genebank is implementing the NIAS Genebank Project to preserve and document plant, microorganism, and animal genetic resources related to agriculture in Japan; however, it lacks a classification of plant gene functions [31]. The PathoPlant database has been developed to explain the molecular processes involved in signal transduction during plant pathogenesis and the interactions between plants and pathogens at the organism level [32]. The Pathogen-Host Interactions database (PHI-base) was established in the year 2005, and PHI-base entries include experimentally verified pathogenicity, virulence, and effector genes from fungal and bacterial pathogens of animal, plant, fungal, and other hosts [33]. The identification and analysis of host-pathogen interactions (HPI) are crucial to study infectious diseases. HPIDB 3.0 is a resource that helps to annotate, predict, and display host-pathogen interactions [34]. Viral infections often cause diseases by disturbing several cellular processes in the infected host. VirusMentha is a new resource for studying virus-virus and virus-host interactions based on integration techniques created for mentha, as well as the detailed curation protocols of the IMEx consortium [35]. An extensive database for predicting Penicillium-crop protein-protein interactions is PCPPI [36]. Currently, data can be amplified by extracting the information from microorganism genomes databases, but there is still a need for more extensive plant pathogen genome databases to understand the mechanism of disease resistance [37].

Identification and Isolation of Resistance (R) genes and Plant NLRs
Gene cloning is improving our understanding of the molecular mechanisms underlying plant-pathogen interactions. Map-based cloning or Positional cloning utilizes the knowledge of genetic map positions. It is the standard method to isolate genes when the phenotype and genomic locations are known. The first cloned R gene was Hm1 from Z. mays against the HC toxin (the host-selective toxin pathogen) secreted by the fungus Cochliobolus carbonum [15]. Gene Hm1 encodes a reductase enzyme that detoxifies the HC toxin and develops resistance in plants against C. carbonum followed by Pto (encoding a serine-threonine kinase) from S. lycopersicum, which confers resistance against Pseudomonas syringae pv. tomato [38]. Most isolated R genes encode cytoplasmic proteins consisting of a central nucleotide-binding site (NBS) domain and a C-terminal domain containing leucine-rich repeats (LRRs), including Cf-9, a predicted membrane protein with an extracellular LRR domain [10]. The Cf-9 gene was isolated from S. lycopersicon through transposon tagging using the Maize Activator/Dissociation (Ac/Ds) system. Similarly, the N gene was isolated from tobacco (N. tabacum) via transposon tagging, and it conferred resistance to Tobacco mosaic virus (TMV) [8]. Furthermore, two genes (RPS2 and RPM1) were isolated from A. thaliana conferring resistance against P. syringae using a map-based cloning approach [39,40], in addition to the L6 gene in flax conferring against Melampsora lini using the Maize Activator/Dissociation (Ac/Ds) system [41]. Due to advancements in plant genomics and genetic engineering techniques, the positional cloning approach has made it easier to clone R genes from various crops or their wild relatives and transfer them into elite breeding lines or cultivars.
Virus infections are also prevalent in maize-growing regions around the world. Maize rough dwarf disease (MRDD) is caused by various species of viruses belonging to the genus Fijivirus. The Rab GDP dissociation inhibitor alpha (RabGDIα) is the host susceptibility factor for rice black-streaked dwarf virus [111]. These resistance alleles are valuable to improve resistance to rough dwarf disease in maize and potentially develop resistance against rice black-streaked dwarf virus in other crops. Sugarcane mosaic virus (SCMV) is one of the severe viral diseases in maize. Two resistance loci, namely Scmv1 and Scmv2, conferring complete resistance against SCMV have been identified. Scmv1 encodes ZmTrxh, a molecular chaperone suppressing viral RNA accumulation in the cytoplasm without stimulating a salicylic acid-or jasmonic acid-mediated defense response [118,119] (Table 5).

Resistance (R) Genes in Arabidopsis (A. thaliana)
The cloning of resistance genes facilitates the development of resistant cultivars and develops an understanding of the evolutionary history of R genes. Most of the R genes identified in Arabidopsis belong to either the TIR-NBS-LRR or LZ-NBS-LRR subclass. In addition, receptor-like kinases (RLKs) are also involved in plant development and defense. The most well-known RLKs in Arabidopsis are the leucine-rich repeat receptor kinases flagellin-sensitive 2 (FLS2) and BAK1, which initiate the MAP kinase cascade upon flg22 recognition, leading to plant innate immunity [120,121]. The TIR-NBS-LRR subclass is defined by an N-terminal region that resembles the cytoplasmic domain of the Toll and interleukin1 transmembrane receptors (TIRs), e.g., RPP1, RPP4, and RPP5 confer resistance to Peronospora parasitica [122][123][124]. In contrast, the LZ-NBS-LRR subclass contains a leucine zipper-like motif (LZ) in place of the TIR domain, e.g., RPS2, RPM1, RPP8, and RPP13 genes confer resistance to P. syringae [39,40,125,126]. Some R genes, RPW7 and RPW8, encode proteins with motifs for a nucleotide-binding site (NBS), and an LRR confers resistance to the powdery mildew pathogens Erysiphe cruciferarum [127].
RPP4-mediated resistance has been associated with multiple defense-signaling components, including EDS1 (enhanced disease susceptibility 1 [128], NDR1 (non-race-specific disease resistance 1) [129], and PBS1 [130], and the absence of functional alleles of either EDS1 or NDR1 leads to enhanced susceptibility to a diverse range of pathogens. In addition, EDS1 is required for RPS4-mediated disease resistance against P. syringae pv. tomato and does not specify resistance to P. parasitica, unlike other EDS1-dependent R genes [131]. The mapping and characterization of the RCH2 locus identified the pair of neighboring genes, namely RRS1 and RPS4, which confer dual resistance against fungal (Colletotrichum higginsianum) and bacterial (Ralstonia solanacearum) pathogens [132,133]. Similarly, map-based cloning has facilitated characterization of the RFO locus (RESISTANCE TO FUSARIUM OXYSPORUM (RFO), which is identical to WAKL22 (WALL-ASSOCIATED KINASE-LIKE KINASE 22) in Arabidopsis [134]. RPS5 belongs to the NBS-LRR subclass, and cloning RPS5 genes has facilitated the characterization of two rps5 mutations. The rps5-1 mutation causes a glutamate-to-lysine substitution within the LRR region and affects the function of several R genes and confers resistance to both pathogens (P. parasitica and P. syringae) [135]. In Arabidopsis, members of both subclasses (TIR-NBS-LRR and LZ-NBS-LRR) confer resistance to the fungus P. parasitica and the bacterium P. syringae, whereas RCY1, belonging to CC-NB-LRR subclass, confers viral resistance. Cucumber mosaic virus (CMV) has the widest host range and causes catastrophic crop loss in many areas. RCY1 is the dominant locus that confers resistance to the yellow strain of ecotype C24 in Arabidopsis [136] (Table 6).

NLR-Parser
NLR-Parser is a tool to rapidly annotate the NLR (nucleotide-binding leucine-rich repeat) complement from sequenced plant genomes. It is a Java application used for the identification of NLR-like sequences. The pipeline was designed to use the MAST output from six-frame translated amino acid sequences and filters for predefined biologically curated motif compositions. Input reads can be derived from, for example, raw longread sequencing data or contigs and scaffolds originating from plant genome projects. The output is a tab-separated file with information on the start and frame of the first NLR-specific motif, whether the identified sequence is a TNL or CNL, and whether it is potentially complete or fragmented. In addition, the output of the NB-ARC domain sequence can directly be used for phylogenetic analyses. NLR-parser can also discriminate pseudogenes by looking for the complete set of motifs defining an NLR protein. It uses motif alignment and a search tool (MAST) to search for 20 conserved motifs found in NLRs that use the highly-specific amino acid motif composition found in plant NLR gene products [148]. It can be downloaded from Git-Hub using the website (https://github. com/steuernb/NLR-Parser, accessed on 3 May 2023).

NLR-Annotator
NLR-Annotator is an extension of NLR-Parser to annotate NLR loci in genomic sequence data. Our pipeline dissects genomic sequences into overlapping fragments, and each fragment is translated in all six reading frames using NLR-Parser to preselect those fragments potentially harboring NLR loci. Using this approach, they could find putative candidate genes for NLR loci in stem rust, leaf rust, powdery mildew, and yellow rust resistance genes [44]. In 2018, NLR-Annotator, the improved version of NLR prediction, was released (https://github.com/steuernb/NLR-Annotator, accessed on 3 May 2023).

NLGenomeSweeper
Another pipeline to annotate functional NLR disease-resistance genes in genome assemblies is NLGenomeSweeper. It is a pipeline that searches a genome for NBS-LRR (NLR) disease-resistance genes based on the presence of the NB-ARC domain. This procedure can be used with a customized NB-ARC HMM consensus protein sequence(s) created for a species of interest for each type of NBS-LRR (TNLs, CNLs, and NLs) and merge them into a single fasta file for use. This pipeline shows high specificity for complete genes and structurally complete pseudogenes. This pipeline identified 152 potential NBS-LRR proteins; 140 of these matched the manually annotated Arabidopsis NLR set, which contains 146 genes (96% sensitivity) [149].

DRAGO2
Disease Resistance Analysis and Gene Orthology (DRAGO 2) is the second version of a pipeline to annotate resistance genes. It is an extensive, freely accessible, and user-friendly online platform for analyzing and predicting plant disease-resistance genes. The input of DRAGO 2 can be either DNA or protein sequences in FASTA format. DRAGO2 is available in PRGdb (http://prgdb.org, accessed on 3 May 2023). The core of the DRAGO2 pipeline is a Perl script that predicts putative pathogen receptor genes (PRGs) and LRR, kinase, NBS, and TIR domains. It can also detect CC and TM domains using COILS 2.2 and TMHMM 2.0c programs. More than 1700 possible PRGs were predicted using the DRAGO2 tools, which have the highest sensitivity compared to other tools [150].

NLRtracker
NLRtracker has been designed to overcome the limitation associated with the existing NLR tools. NLRtracker uses InterProScan and the predefined NLR motifs to annotate all sequences in a given proteome or transcriptome and then extracts and annotates NLRs based on the core NLR sequence features (late blight R1, TIR, RPW8, CC, NB-ARC, LRR, and integrated domains) found in the RefPlantNLR dataset. Additionally, NLRtracker extracts the NB-ARC domain for a comparative phylogenetic analysis [151].

CRISPR Gene Editing for the Generation of Disease Resistance
The CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas9 (CRISPRassociated) system has surpassed alternative genome editing methods due to its simplicity, flexibility, better success rate, and cost-effectiveness. The CRISPR/Cas9 system can efficiently introduce mutations, including INDELs (insertion mutations and deletion) and base substitutions in the target site. One significant advantage of using the CRISPR/Cas9 system is the ability to edit multiple target genes simultaneously [152]. Several efficient plant genome editing web-based tools are available for designing sgRNAs and analyzing post-genome editing data [153] (Table 8). CRISPR/Cas systems have been divided into six types based on their signature Cas genes. Class 1 CRISPR/Cas systems (types I, III, and IV) employ multi-Cas protein complexes for interference, whereas class 2 systems (types II, V, and VI) accomplish interference with single effector proteins in complex with CRISPR RNAs (crRNAs) [154]. This system has been successfully applied to various plant species, such as A. thaliana, O. sativa, N. tabacum, S. bicolor, T. aestivum, Z. mays, G. max, S. lycopersicum, S. tuberosum, P. alba, M. domestica, and Musa species, to combat viral infection and fungal and bacterial diseases [26,155]. There are several strategies for developing plant disease resistance via the CRISPR/Cas system [156]: (i) knock-out of susceptibility genes of disease (e.g., MLO; a mildew resistance locus O) [27], (ii) deletion or modification of cis-elements in promoters [157], (iii) modification of the amino acid sequence of surface receptor proteins to suppress secreted pathogen effectors [153], (iv) knockdown of negative regulators of plant immunity [158], and (v) modification of central regulators of the defense response [159].
The CRISPR/Cas9 system has facilitated efficient and precise targeted mutagenesis in plants to enhance resistance to fungal diseases. Mildew resistance locus O (MLO) is the most widely studied gene for resistance to fungal diseases. Wild-type alleles of MILDEW RESISTANT LOCUS O (Mlo) are conserved throughout monocots and dicots, conferring susceptibility to the powdery mildew fungi Oidium neolycopersici. The generation of a resistant variety using CRISPR/Cas9 technology against the powdery mildew pathogen was reported in various crops: H. vulgare, A. thaliana, S. lycopersicum, Pisum sativum, Fragaria vesca, Capsicum annuum, T. aestivum, C. sativus, Rosa hybrid, N. tabacum, C. melo, V. vinifera, and M. domestica [27]. SlMlo1 is a major gene responsible for powdery mildew disease in S. lycopersicum, among 16 MLO genes studied so far. CRISPR/Cas9 technology has been employed to knock out SlMlo1 in developing resistance against the powdery mildew fungus O. neolycopersici without affecting the phenotype [27]. Similarly, a CRISPR/Cas9 system was used to mutate the susceptibility gene of Powdery Mildew Resistance 4 (PMR4), resulting in resistance to O. neolycopersici in S. lycopersicum. Additionally, both TALENs and CRISPR tools have been used to introduce mutations in one (TaMLO-A1) of the three MLO homoalleles, which resulted in improved resistance against B. graminis f. sp. tritici infection in T. aestivum [160,161]. In a similar study, a CRISPRmediated MLO mutation resulted in the development of resistance to powdery mildew in H. vulgare (B. graminis f. sp. hordei), but at the same time, it increased susceptibility to the blast fungus M. grisea (M. oryzae) in O. sativa [162]. The CRISPR/Cas9-mediated editing of two susceptible genes, MLO-6 and DMR, resulted in increased resistance against the powdery mildew fungus Erysiphe necator and downy mildew fungus Plasmopara viticola in V. vinifera [163]. Another study in V. vinifera demonstrated that loss of the VvMLO7 gene increased resistance against E. necator through gene knock-down [164,165]. The CRISPR/Cas9-mediated knock-out of two genes, Solyc08g075770 and SlymiR482e-3p, in the different studies, resulted in resistance against the pathogen that causes Fusarium wilt in S. lycopersicon [166,167]. Similarly, a mutation in the Clpsk1 gene enhanced resistance against F. oxysporum in C. lanatus [168]. EDR1 (enhanced disease resistance) is highly conserved across plant species and negatively affects plant immunity. In Arabidopsis, EDR1 was reported to be a negative regulator of powdery mildew resistance, and this regulation was mediated by suppressing salicylic acid and enhancing abscisic acid signaling. Three homologs of the TaERD1 gene were mutated using CRISPR/Cas9, and the resultant Taedr1-mutant plants showed a significant reduction in blast lesions and resistance to powdery mildew in T. aestivum [169]. It was reported that the expression of EDR1 was induced by jasmonic acid (JA), salicylic acid, ethylene, and abscisic acid [170]. Moreover, both jasmonic and salicylic acid accumulation is associated with enhanced resistance against X. oryzae pv. oryzae (Xoo) in O. sativa. OsEDR1-knock-out plants demonstrated enhanced resistance against the bacterial blight-causing pathogen Xoo [171]. DMR6 (downy mildew resistance 6) has been identified as a susceptibility gene in S. tuberosum [172] and Arabidopsis [173]. Two DMR genes (StDMR6-1 and StDMR6-2) were edited simultaneously in S. tuberosum resulting in enhanced resistance against the late blight fungus P. infestans [174].
Rice blast is one of the most devastating diseases that affect rice production worldwide. Ethylene-responsive factors (ERFs) of the APETELA2/ERF (AP2/ERF) superfamily play crucial roles in adaptation to various biotic stress. Rice blast resistance to the fungus M. oryzae was enhanced mediated through the CRISPR/Cas9-mediated mutation of ERF922 gene [175]. Knock-down of the AP2/ERF transcription factor reduced abscisic acid accumulation and increased resistance against M. oryzae [176]. Similarly, the CRISPR/Cas9-mediated knockout of AtERF019 in A. thaliana enhanced resistance to Phytophthora parasitica by suppressing PAMP-triggered immunity [177]. The overexpression of defense genes is one of the key biotechnological tools exploited to develop resistance against plant pathogens. In Theobroma cacao, overexpression of the TcNPR1 (Non-expressor of Pathogenesis-Related 1) gene reduced infection caused by Phytophthora spp. in leaf tissue [158].
Microrchidia (MORC) proteins are important nuclear regulators in prokaryotes and eukaryotes, involved in transcriptional gene silencing and the maintenance of genome stability [178]. In Arabidopsis, the role of MORC1 was discovered in plant immunity against turnip crinkle virus (TCV). Moreover, the role of AtMORC1, AtMORC2, and AtMORC6 are reported in multiple layers of defense responses against P. syringae and Hyaloperonospora arabidopsidis [49,179]. The CRISPR-Cas9 system from Streptococcus pyogenes (CRISPR/SpCas9) was used to introduce a mutation at HvMORC1 and HvMORC6a genes in H. vulgare. Similarly, MORCs have also been studied in S. tuberosum (StMORC1), S. lycopersicum (SlMORC1), and Nicotiana benthamiana (NbMORC1) [180,181]. WRKYs (WRKY transcription factors) have been identified in different plants in plant immune responses. Mutant analyses in Arabidopsis have revealed direct links between specific WRKY proteins (WRKY8, WRKY11, WRKY33, WRKY38, WRKY53, WRKY62, and WRKY70) and defense responses against P. syringae. Coronatine (COR) is the phytotoxic compound produced by the pathogen P. syringae pv tomato DC3000 (Pto3000), causing bacterial speck disease in S. lycopersicon. The CRISPR/Cas9-mediated mutation of the S1JAZ2 gene resulted in resistance to bacterial speck disease infestation in S. lycopersicum [182]. The role of the WRKY70 gene in the disease response to the fungus Sclerotinia sclerotiorum in B. napus was also documented in the literature [159]. In a similar study, the CRISPR/Cas9-mediated targeted mutagenesis of VvWRKY52 produced mutant lines in V. vinifera and the knock-out of WRKY52 enhanced resistance to Botrytis cinerea, causing gray mold disease [165].
Many viruses infecting economically important crops belong to the category of RNA viruses. CRISPR/Cas technology has been applied successfully to develop resistant plants against RNA viruses. Rice tungro disease is a severe problem caused by an interaction between rice tungro spherical virus and rice tungro bacilliform virus. In plants, eIF4E and eIF(iso)4E assist in recruiting ribosomes to the 5 UTRs of mRNAs, which is eventually required to translate viral proteins. The copy numbers of the eIF4E and eIF(iso)4E genes vary from species to species [183]. A CRISPR/Cas9-mediated mutation in eIF4G provided resistance to rice tungro streak spherical virus in a susceptible variety (IR64) of O. sativa [184]. Mutation of the recessive eIF4E gene enhanced resistance against turnip mosaic virus in Arabidopsis and cucumber vein yellowing virus in cucumber [185,186]. Similarly, RNA virus resistance has been demonstrated by silencing the eIF4E gene in S. lycopersicum and C. melo [28,29]. A recent discovery of FnCas9 (Cas endonucleases) from Francisella novicida may be used as a new tool for attacking the genome of plant RNA viruses. Fn-Cas9 was used to develop resistance against Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV) in N. benthamiana and Arabidopsis plants, respectively [187]. Characterization of the functionality of Cas13a of Leptotrichia shahii (LshCas13a) demonstrated that the single effector Cas13a protein was a programmable RNA-guided single-stranded RNA (ssRNA) ribonuclease that provided immunity against bacteriophages of the bacteria Escherichia coli [188]. The LshCas13a system was used for developing resistance to Southern rice black-streaked dwarf virus (SRBSDV) and Rice stripe mosaic virus (RSMV) in O. sativa [189].
O. sativa is extensively used for genome editing studies against bacterial disease resistance. Rice bacterial blight is one of the invasive diseases caused by bacterial X. oryzae pv. oryzae (Xoo) [190]. X. oryzae secretes transcription-activator-like effectors (TALes) that bind specific promoter sequences and induce sucrose transporter genes (SWEET11, SWEET13, and SWEET14). The expression of sucrose transporter genes is required for disease susceptibility and mutations in effector binding element (EBE) regions in promoters of SWEET11, SWEET13, and SWEET14 genes [157]. The CRISPR/Cas9-mediated knockout of the Os8N3 gene resulted in enhanced resistance to most Xoo and bacterial blight [191]. Similarly, induced mutations in O. sativa into the coding regions of TMS5 (thermosensitive male sterile), Pi21 (proline-rich protein), and Xa13 (bacterial blight resistance) genes via CRISPR/Cas9 improved resistance against rice blast and bacterial blight [192]. The genus Xanthomonas is one of the significant genera affecting various horticultural crops. Citrus canker is one of the major diseases of citrus caused by the bacterium Xanthomonas citri ssp. citri (Xcc). Lateral Organ Boundaries 1 (CsLOB1) is a transcription factor that assists in the proliferation of X. citri spp. citri (Xcc). Effector binding element (EBE) regions present in the CsLOB1 promoter are recognized by the Xcc effector (PthA4), and expression of the CsLOB1 gene facilitates canker development in Citrus sp. CRISPR/Cas9-mediated editing of EBEs in the CsLOB1 promoter and coding region of the CsLOB1 gene provides resistance to citrus canker in C. sinensis and C. paradise [193]. Similarly, another transcription factor, WRKY22, was mutated through CRISPR/Cas9 technology and resulted in resistance to citrus canker in C. sinensis [194]. Fire blight is another devastating disease caused by Erwinia amylovora in M. domestica. The CRISPR/Cas9-mediated mutation of disease-specific interacting protein (DIPM-1, DIPM-2, and DIPM-4) genes provides resistance to the golden delicious variety of M. domestica against fire bight disease [195]. The application of the CRISPR/Cas system for disease resistance development by either targeting the pathogen genome or host genes to interfere with susceptibility has become more effective due to its simple operation, good knockout effect, low cytotoxicity, high specificity, and universal applicability.
The CRISPR system has attracted more and more attention because CRISPR/Cas-induced mutations create pathogen-resistant genotypes when resistance resources in natural populations or wild relatives are limited. CRISPR/Cas also offers the opportunity to develop designer plants with multiple valuable attributes and resistance against biotic and abiotic stress. Thus, this technology should be explored and improved for creating novel diseaseresistance genes/genotypes, which ultimately need reduced pesticide applications. These developments in genome editing will undoubtedly be advantageous for environmentally sustainable agriculture.
Intracellular nucleotide-binding leucine-rich repeat (NLR) receptors recognize pathogen effectors and initiate the immune response. The mechanisms of plant NLR activation remain unresolved, whereas animal NLRs undergo oligomerization upon binding to their effectors to activate downstream signaling. Our understanding of the plant NLR activation process has greatly increased due to the available structural data of CNL and TNL resistosomes. The composition and three-dimensional CNL structures of an Arabidopsis ZAR1 (HopZ-activated resistance) using cryo-EM microscopy structures illustrate differences between inactive and intermediate states of ZAR1 [196]. Similar studies uncovered the CNL structure of wheat Sr35 and found its resemblance to the ZAR1 resistosome structure of Arabidopsis [83,197]. In addition, the cryo-EM structures of TNL resistosomes from RPP1 (recognition of Peronospora parasitica 1) and ROQ1 (recognition of Xanthomonas outer protein Q 1) from A. thaliana and N. benthamiana, respectively, were determined using cryo-EM microscopy [198,199]. Recent advancements in computational methods, such as AlphaFold, have been used to predict the three-dimensional structure of the protein AVRamr1 (recognition of P. infestans effector) [200]. This structural framework moves us closer to developing novel immune receptors with modified recognition specificities and more effective plant disease-resistance proteins. Modern technology recognizes potential target regions of NLRs and the conserved resistosome structure, highlighting the future possibility of crop improvement through structure-guided NLR engineering. However, some questions are yet to be answered, such as whether all CNL and TNL immune receptors exhibit resistosome properties or if NLR activation requires the resistosome, as well as the possibility of monitoring resistosome formation using engineered NLR chimera.

Conclusions
NLRs play a crucial role in plant immunity by activating the strong resistance response leading to plant disease resistance. NLRs have a central nucleotide-binding (NB) domain which acts as an on/off activation switch, followed by a leucine-rich repeat (LRR) domain. The structure diversity, abundance, and chromosomal distribution of NLRs are fundamental for understanding disease resistance. The availability of high-throughput sequencing technology allows for the identification and cloning of several candidate resistance genes in different plant species. Gene editing technologies create a novel variation at the gene and genome levels. However, pathogens can eventually overcome disease resistance based on single-base editing due to their rapid evolution and genetic diversity of bacterial and fungal populations. The advanced variants of genome-editing tools, such as CRISPR/Cas, have brought many insights into the molecular mechanisms of site-specific mutagenesis. Moreover, durable resistance can be produced by pyramiding numerous genes and/or altering the plant and pathogen genomes using CRISPR/Cas9 technology. Protein engineering has redefined our ability to develop new or improved molecular recognition capabilities of NLRs, and engineered intracellular immune receptors can potentially improve disease resistance. The research on NLR proteins has been limited due to the unavailability of adequate three-dimensional structures of individual domains and homology models. However, in recent years, a significant advance in cryo-electron microscopy resolved the full-protein cryo-EM structure of NLR complexes, providing comprehensive insights into the complex biological mechanisms and functional complexity of NLRs. Moreover, modern computational technology, such as Alphafold, ca predict the three-dimensional structures of proteins with higher accuracy. These cutting-edge technologies may generate designer NLR receptors to confer broad-spectrum resistance in crop plants. Furthermore, more comprehensive tools are required for understanding accurate protein structures, ligand binding, and host-pathogen interactions. Overall, integrated computational and molecular biology tools provide a practical approach for efficiently breeding multiline cultivars and a strategy for generating designer crops with broader resistance and high yields.