R gene-mediated resistance in plant diseases PDF

Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23 https://doi.org/10.1007/s13562-023-00858-w REVIEW ARTICLE R gene-mediated resistance in the management of plant diseases Aditi Tailor1 · Satish C. Bhatla2 Received: 14 March 2023 / Accepted: 11 October 2023 / Publ...

Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23 https://doi.org/10.1007/s13562-023-00858-w REVIEW ARTICLE R gene-mediated resistance in the management of plant diseases Aditi Tailor1 · Satish C. Bhatla2 Received: 14 March 2023 / Accepted: 11 October 2023 / Published online: 8 November 2023 © The Author(s), under exclusive licence to Society for Plant Biochemistry and Biotechnology 2023 Abstract Biotic stress factors cause massive damage to crops and account for major economic losses globally. With the global tem- perature on the rise, a great threat looms on the global economy and food security as sustainable agriculture is expected to be devastated by larger outbreaks of diseases worldwide. Plants have developed versatile counter-strategies to overcome these stressors. These strategies rely on preformed defense or induced defense responses which, in turn, entail a complex crosstalk among different signaling pathways. Once the presence of a pathogen is detected, the plant host switches on its defense response. Very specific recognition of the invading pathogen is mediated via the detection of their Avr effector pro- teins by the cognate elements in the plant host, i.e., the R proteins, encoded by R genes. Successful recognition of Avr by the cognate host R proteins sets distinct signaling cascade(s) into motion to activate defense responses. The nucleotide-binding leucine-rich repeat proteins (NB-LRRs or NLRs) represent the major class of R proteins and insights gained regarding their structural complexity and underlying signal transduction has enabled researchers to exploit them for competent introgression of disease resistance in plants against a single or a broad range of pathogens. In addition to conventional breeding, more recent biotechnological interventions have also been employed to develop plants with improved disease resistance with the use of R genes. The domain of R gene-mediated disease resistance has gained much attention from plant breeders and through experimental interventions, many promising opportunities for conferring resistance for effective management of diseases have come forth. This review attempts to summarize the progress made hitherto and focuses on how the knowledge base so created has been used to find out solutions to the major biotic problems occurring throughout the world. Keywords Disease resistance · R genes · Breeding · Marker assisted selection · Transgenics Introduction (Savary et al. 2019). With global temperatures on the rise, larger disease outbreaks are anticipated worldwide which The agriculture sector accrues huge losses due to biotic will be an impediment to the goal of achieving sustainable stress factors, including a wide array of pests and pathogens, agriculture and, thus pose a great threat to food security with a reduction to the tune of up to 40% in the annual agro- (Zayan 2019). This, in turn, presses upon the need to look nomic harvest in many severe cases (Savary et al. 2019). It for measures to improve the tolerance of plants to biotic has been estimated that to feed the estimated 10 billion peo- stress factors. ple by 2050, the food production will have to be increased by Over years of evolution, plants have come to develop 60% (FAO 2019). Meeting this ambitious target will require efficient mechanisms to detect the presence of pathogens not only an increase in production but a simultaneous reduc- and subsequently deploy their defense to eliminate the biotic tion in the crop losses caused by various pests and pathogens threat (Dodds and Rathjen 2010; Couto and Zipfel 2016; Jones et al. 2016). The plant defense works in two layers, i.e., the pathogen-associated molecular patterns (PAMPs) trig- * Satish C. Bhatla gered immunity (PTI) and effector-triggered immunity (ETI) [email protected] (Dodds and Rathjen 2010; Jones et al. 2016). PTI forms the 1 Scientist B, Genetics and Tree Improvement Division, first level of surveillance, allowing detection of conserved ICFRE-Arid Forest Research Institute, Jodhpur, pathogen-associated molecules (or PAMPs) by the plant cell Rajasthan 342005, India surface receptors, i.e., pattern recognition receptors (PRRs), 2 Department of Botany, University of Delhi, Delhi 110007, followed by activation of a basal level response (Zipfel 2014; India 13 Vol.:(0123456789) 6 Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23 Saijo et al. 2018). However, pathogens can bypass the induc- 2014; Le Roux et al. 2015; Maqbool et al. 2015; Adachi tion of PTI by directly delivering “effectors” (encoded by et al. 2019). A successful R-Avr recognition leads to the Avr genes) into the cytoplasm (Zipfel 2014; Toruño et al. initiation of a signaling cascade by NLRs to accomplish 2016). As a countermeasure, plants are capable of specifi- HR, which may include signaling components like helper cally recognizing these effectors and eliciting a second, and NLRs [Activated Disease Resistance gene 1 (ADR1) and much stronger, level of defense response, i.e., the ETI, often N-Required Gene 1 (NRG1)], mitogen-activated protein culminating in a hypersensitive response (HR) (Jones et al. kinases (MAPKs), ­Ca2+ and phytohormones, like SA, and 2016). This specific recognition of pathogen-derived effec- transcription factors (TFs), leading to activation of ETI and tors is mediated by specialized intracellular receptors or R cell death, and activation of expression of defense response proteins encoded by R genes (Jones et al. 2016). genes (Cui et al. 2015; Jones et al. 2016; Zhang and Li 2019; Plants host large numbers of R genes for providing resist- Sun et al. 2021; Fig. 1). The TNLs can activate either EDS1/ ance against a wide array of pathogens (Kourelis and van der PAD4/ADR1 (Enhanced Disease Sensitivity 1/Phytoalexin Hoorn 2018; Kourelis et al. 2021; Table 1). The R proteins Deficient 4/Activated Disease Resistance gene 1) and EDS1/ not only function as putative receptors for pathogen detec- SAG101/NRG1 (Enhanced Disease Sensitivity 1/Senescence tion but also activate downstream signaling cascades to trig- Associated Gene 101/N-Required Gene 1) signaling com- ger defense response. The nucleotide-binding leucine-rich plexes while CNLs activate downstream component NDR1 repeat (NB-LRR or NLR) class represents the largest repre- (Non-race specific Disease Resistance 1) (Century et al. sentative group of R proteins (Kourelis and van der Hoorn 1995; Knepper et al. 2011a, b; Wagner et al. 2013; Castel 2018; Kourelis et al. 2021). Structurally, NLRs are multid- et al. 2019; Lapin et al. 2019; Wu et al. 2019; Saile et al. omain proteins containing conserved domains, i.e., an N-ter- 2020; Dongus and Parker 2021; Sun et al. 2021). NLRs also minal (TIR/CC/RPW8), a central nucleotide-binding (NB) interact with the HSP90/RAR1/SGT1 (Heat shock protein domain (containing nucleotide-binding site or NBS), and 90/ Required for Mla12 Resistance 1/Suppressor of G-two the C-terminal leucine-rich repeats (LRR) domain, (Cesari allele of skp1) chaperone complex which regulates the accu- 2018; Kourelis and van der Hoorn 2018). The N-terminal mulation of NLRs, and their maturation and stability (Seo CC and TIR domains are believed to be involved in activat- et al. 2008; Shirasu 2009). SGT1 with the help of SKP/CUL/ ing signal transduction pathways and facilitating homodi- F-box or SCF (Suppressor of G-two allele of skp1/CULLIN/ merization and formation of heterocomplexes via interaction F-box) E3 ubiquitin ligase complex, and proteins acting as with different NLRs as well as oligomerization with other repressors of host defense via ubiquitination and subsequent proteins (Maekawa et al. 2011; Nishimura et al. 2017; Zhang degradation by 26 S proteasome (Tör et al. 2003). This SCF et al. 2017). The central NB domain is crucial for NLRs to complex also regulates R protein accumulation negatively undergo conformational alterations and to facilitate inter- when the defense is not required (Cheng and Li 2012). Some domain and intermolecular interactions within and between CNLs and helper NLRs also oligomerize into pentameric NLRs (Takken et and Goverse 2012; Wang et al. 2019a; Ma structures called ‘resistosome’ and become integrated into et al. 2020; Martin et al. 2020). The C-terminal LRR domain the plasma membrane where they aid in C ­ a2+ influx required displays a high degree of variability and is responsible for for activating cell death by forming ­Ca2+ permeable chan- the recognition of a large variety of effector molecules via nels (Wang et al. 2019a, b; Bi et al. 2021). SA accumulation protein-protein interactions (Parniske et al. 1997; Ellis et al. in the cytoplasm triggers redox state change, thus facilitating 1999). monomerization of NPR1 (Nonexpressor of PR Genes 1), R proteins employ distinct mechanisms to detect specific the key regulator of SA-mediated responses, by triggering pathogen effectors through direct or indirect modes (Cesari the disulfide bond breakdown and assisting in its separa- 2018). Some R proteins can directly perceive cognate path- tion from NPR1 oligomeric complex (Tada et al. 2008; Wu ogen effectors by means of physical interaction, such as et al. 2012). NPR1 monomers can translocate to the nucleus, those encoded by the flax L567 locus conferring tolerance recruits TFs, and activate the expression of defense response to Melampsora lini (Flor 1971; Ellis et al. 1999; Ravensdale genes. et al. 2010). Others recognize effectors indirectly by moni- A wide array of R genes has been described to show toring the effector-induced modifications of host target pro- effectiveness against a variety of pathogens and thus, R gene teins known as “guardees” (Guard Model) or by employing research presents a promising outlook for benefitting the the assistance of decoy proteins (Decoy model) that share agriculture sector by disease management (Kourelis and van similarities with the guardee proteins (Cesari 2018; Adachi der Hoorn 2018; Table 2). For reducing damage by biotic et al. 2019). In another indirect model, the integrated decoy stress agents and in turn, enhance crop production, plants model, NLRs show the presence of atypical integrated breeders have been utilizing R genes for conferring disease domains (IDs) mimicking the effector targets and working resistance as well as broadening of disease resistance spec- in complementary pairs to activate defense (Cesari et al. trum to various pathogens of major crop species (Pandolfi 13  Table 1  Examples of pathogen effectors, their host targets, and corresponding R proteins/genes Effector Organism Target Host Mode of action of effector Corresponding References R protein/gene Bacterial pathogens PopP2 Ralstonia solanacearum EDS1, PAD4 Arabidopsis Acyltransferase activity RPS4/RRS1-R Huh ( 2021) thaliana AvrRpm1 Pseudomonas syringae RIN4 A. thaliana ADP-ribosyltransferase RPM1 Mackey et al. (2002) activity AvrPphB1 P. syringae BIK1, PBS1 A. thaliana Protease activity RPS5 Shao et al. (2003), Lu et al. (2010) AvrRps4 P. syringae (-) A. thaliana (-) RPS4 Sohn et al. (2012) AvrB, P. syringae pv. tomato RIN4 A. thaliana Indirect phosphorylation by RPM1 Mackey et al. (2002) AvrRpt2 P. syringae RIN4 A. thaliana Cysteine Protease activity RPS2 Mackey et al. (2003), Axtell and Staskawicz (2003) Avr-Pto and AvrPtoB P. syringae pv. FLS2, BAK1, CERK1, Fen Solanum lycopersicum E3 ubiquitin ligase, degrada- Pto (decoy)/Prf Gutierrez et al. (2010) tomato and Pto tion of FLS2, BAK1, CERK1, Fen and Pto HopF1r P. syringae ZRK3 A. thaliana ADP-ribosyltransferase ZAR1 Seto et al. (2017) HopZ1a P. syringae ZED1 A. thaliana Acetyltransferase ZAR1 Lewis et al. (2013) AvrAC Xanthomonas campestris BIK1, PBL1 A. thaliana Uridine 5’-monophosphate RKS1/ZAR1 Wang et al. (2015) transferase activity AvrBs2 X. campestris (-) Capsicum annuum Glycerophosphoryl diester Bs2 Zhao et al. 2011 Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23 phosphodiesterase RaxX X. oryzae pv. oryzae (-) Oryza sativa Promotes root growth by Xa21 Pruitt et al. 2015 mimicking the plant pep- tide hormone PSY (plant peptide containing sulfated tyrosine) Fungal pathogens ATR2 Hyaloperonospora arabidi- (-) A. thaliana (-) RPP2A/RPP2B Sinapidou et al. 2004 opsis Avr2 Phytophthora infestans BSL1 Solanum demissum Interacts with BSL2 R2 Saunders et al. 2012 Avr2 P. infestans BSL2, BSL3 Solanum mochiquense Interacts with BSL2/3 Rpi-mcq1 Wang et al. 2021 Avrblb2 P. infestans C14 (papain-like cysteine Solanum bulbocastanum Prevents secretion of C14 Rpi-blb2 Bozkurt et al. 2011 protease) Avr4 Cladosporium fulvum (-) S. lycopersicum Chitin binding protein, Cf-4 van den Burg et al. 2006; van protection of chitin against Esse et al. 2007 hydrolysis by plant chi- tinases Avr2 C. fulvum PIP1 and RCR3 S. lycopersicum Protease inhibitor Cf2 Shabab et al. 2008; van Esse et al. 2008 13 7  8 Table 1  (continued) Effector Organism Target Host Mode of action of effector Corresponding References R protein/gene 13 AvrMla Blumeria graminis f. sp. (-) Hordeum vulgarae (-) Mla6 Halterman et al. 2001 hordei AvrL Melamspora lini (-) Linum usitatissimum (-) L Dodds et al. 2004 AvrPita Magnoporthe oryzae COX11 O. sativa Interaction with COX and Pita Han et al. 2021 increase in its activity, causing decreased ROS accumulation triggered by fungal chitin Avr2 Fusarium oxysporium f. sp. (-) S. lycopersicum Attenuation of immunity I2 Houterman et al. 2009; Ma lycopersici et al. 2013 AvrFom2 F. oxysporum f. sp. melonis (-) Cucumis melo (-) Fom2 Brotman et al. 2013; Schmidt et al. 2016 Viral pathogens Coat protein (CP) Potato virus X (PVX) Solanum tuberosum Rx1 Richard et al. 2021 TMV replicase heli- Tobacco mosaic virus Nicotiana tabacum N Erickson et al. 1999 case fragment(p50) (TMV) CP Tobacco mosaic virus C. annuum L locus Tomita et al. 2011 (TMV) Prv2 Papaya ring-spot virus C. melo Prv Brotman et al. 2013 (PRSV) UN Turnip mosaic virus Brassica campestris ssp. TuR3, TuRB07 Ma et al. 2010; Jin et al. 2014 (TuMV) chinensis Makino cv. Duanbaigeng CP Cucumber mosaic virus, yel- A. thaliana RCY1 Takahashi et al. 2012 low strain (CMV-Y) CP Potato virus Y (PVY) Solanum stoloniferum Rysto Grech-Baran et al. 2020 Rep/C1 Tomato yellow leaf curl Solanum habrochites Ty2 Shen et al. 2020 virus (ToYLCV) NSm Tomato spotted wilt virus Solanum alata RTSW Huang et al. 2018 (TSWV) BAK1- Brassinosteroid-associated kinase 1; BIK1- Arabidopsis Botrytis-induced kinase 1; BSL1- BSU-like protein 1 (BSU -bri1 SUPPRESSOR1; BRI1- Brassinosteroid (BR) receptor); C14- Papain-like cysteine protease 14; CERK1- Chitin elicitor receptor kinase 1; CMV-Y- Cucumber mosaic virus, yellow strain; COX11- Cytochrome C oxidase subunit 11; CP- Coat protein; EDS1- Enhanced Disease Sensitivity 1; FLS2- Flagellin sensing 2; NSm- Non-structural movement protein; p50- TMV replicase helicase fragment; PAD4- Phytoalexin deficient 4; PBL1- AvrPPHB susceptible 1-like 1; PBS1- Arabidopsis AvrPPHB susceptible 1; PIP1- Phytophthora inhibited protease 1; PRSV- Papaya ring-spot virus; PVX- Potato virus X; PVY- Potato virus Y; RCR3- Required for Cladosporium resistance 3; RCY1- Resistance to the yellow strain of cucumber mosaic virus 1; Rep/C1- Replication associated protein; RIN4- RPM1-interacting protein 4; RPM1- Resistance to Pseudomonas syringae pv. maculicola 1; RPP2A/RPP2B- Recognition of Peronospora parasitica 2 A/ Recognition of Peronospora parasitica 2B; RPS2- Resistance to Pseudomonas syringae 2; RPS4/RRS1-R Resistance to P. syringae 2/Resistance to Ralstonia solanacearum 1-Resistant; RPS5- Resistance to Pseudomonas syringae 5; RRS1- Resistance to Ralstonia solanacearum 1; RTSW- Resistance to Tomato spotted wilt virus; TMV- Tobacco mosaic virus; ToYLCV- Tomato yellow leaf curl virus; TSWV- Tomato spotted wilt virus; TuMV- Turnip mosaic virus; ZAR1- Hopz-activated resistance 1; ZED1- Hopz-ETI-deficient 1; ZRK1- ZED1-related kinase 1 (aka RKS1); ZRK3- ZED1-related kinase 3; (-) Not reported Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23 Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23 9 Fig. 1  Pathogen-derived effectors are perceived by NLRs, CNL, and activate the expression of defense response genes. Some NLRs and TNL. Upon successful effector recognition, NLRs trigger sign- bear a domain resembling WRKY TF and can activate the defense aling, including helper NLRs (ADR1 and NRG1), C ­ a2+, and phyto- genes. [Abbreviations: ADR1- Activated disease resistance gene 1); hormones, like SA, leading to activation of ETI and cell death and CDK8- Cyclin-dependent kinase 8; CNL- Coiled-coil (CC) nucleo- defense response genes. The TNLs activate two signaling modules, tide-binding leucine-rich repeat (CC-NB-LRR or CC-NLR); E2- E2 EDS1/PAD4/ADR1 and EDS1/SAG101/NRG1, and CNLs activate conjugating enzyme; EDS1- Enhanced disease sensitivity 1; HSP90- NDR1. NLRs also interact with HSP90/RAR1/SGT1 complex which Heat shock protein 90; NDR1- Non-race specific Disease Resist- regulates the NLR accumulation and stability. Also, with the help of ance 1; NPR1- Nonexpressor of PR Genes 1; NRG1- N-required the SKP/CUL/F-box (or SCF) ubiquitin ligase complex, a target pro- gene 1; PAD4- Phytoalexin deficient 4; RAR1- Required for Mla12 tein that acts as a repressor of host defense can be targeted by ubiqui- Resistance 1; RBX- Ring-box protein 1; ROS- Reactive oxygen spe- tination for subsequent degradation by 26 S proteasome. Some CNLs cies; SAG101- Senescence associated gene 101; SCF- Suppressor of and helper NLRs also oligomerize into pentameric structures called G-two allele of skp1/CULLIN 1/F-box; SGT1- Suppressor of G-two ‘resistosome’ that aid in ­Ca2+ influx required for activating cell death. allele of skp1; T- Target protein; TNL- Toll/interleukin 1 receptor SA accumulation in the cytoplasm triggers redox state change, thus (TIR) nucleotide binding leucine-rich repeat (TIR-NB-LRR or TIR- facilitating monomerization of NPR1 by separation from NPR1 oli- NLR); Ub- Ubiquitin] gomeric complex. NPR1 monomers can translocate to the nucleus et al. 2017; Dong and Ronald 2019; Bentham et al. 2020; van (Dong and Ronald 2019; van Esse et al. 2020; van Wersch Esse et al. 2020; van Wersch et al. 2020a). More recently, the et al. 2020a). R gene pyramiding has also emerged as an advent of transgenic technology has led to the development advantageous strategy for broadening the pathogen spec- of specifically engineered plants having improved resist- trum (Dong and Ronald 2019; van Wersch et al. 2020a). ance in terms of a broader spectrum and enhanced durability Both traditional breeding methods and modern transgenic 13  10 Table 2  Examples of disease resistance developed in plants using various strategies Gene Pathogen/disease Donor Recipient Method References 13 Bs2 Xanthomonas perforans Pepper (C. annuum) Tomato (S. lycopersicum) Genetic transformation Horvath et al. 2012; Kunwar et al. 2018 Pm3b Blumeria graminis f. sp. tritici Wheat Spring wheat (T. Genetic transformation Brunner et al. 2011 aestivum) Pm3 Blumeria graminis f. sp. tritici Wheat Spring wheat cv. Bobwhite Genetic transformation Brunner et al. 2012 Bcl-2 F. oxysporum f. sp. Human (Homo Banana cv. Sukali Genetic transformation Magambo et al. 2016 cubense race 1 sapiens) Ndiizi RPW8.1 and RPW8.2 Powdery mildew (Erysiphe Arabidopsis N. tabacum and N. bentha- Genetic transformation Xiao et al. 2003 orontii, E. cichoracearum, miana and Oidium lycopersici) Ve1 Verticillium Tomato Arabidopsis Genetic transformation Fradin et al. 2011 Rvi6 (formerly HcrVf2) Venturia inaequalis Malus floribunda M. domestica cv. Gala Genetic transformation Vanblaere et al. 2011; 2014 Pi54 and Pi54rh Magnaporthe oryzae Japonica rice cv. Taipei 309 Genetic transformation Kumari et al. 2017 or TP309 Pib, Pi25, and Pi54 Magnaporthe oryzae Rice (Oryza sativa var. Rice cv. Kasalath (var. indica) Genetic transformation Peng et al. 2021 japonica and Oryza sativa and Zhenghan 10 (var. var. indica) japonica) Rpi-blb1 and Rpi-blb3, and P. infestans S. bulbocastanum S. tuberosum cv. ‘Delikat’ Genetic transformation Rakosy-Tican et al. 2020 R3a and R3b Rpi-blb1 and Rpi-blb2 P. infestans S. bulbocastanum cv. ‘Fon- S. tuberosum variety “For- Genetic transformation Storck et al. 2012 tane’ tuna” Rpi-sto1, Rpi-vnt1.1and Rpi- P. infestans S. stoloniferum, S. venturi, S. tuberosum cv. ‘Désirée’ Genetic transformation Zhu et al. 2012; Haesaert et al. blb3 and S. bulbocastanum 2015a RB, Rpi-blb2, and Rpi-vnt1.1 P. infestans S. bulbocastanum and S. S. tuberosum cv. ‘Désirée’ and Genetic transformation Gishlain et al. 2019 venturii ‘Victoria’ Rpi-vnt1.1 and Rpi-sto1 P. infestans S. venturii and S. stoloniferum S. tuberosum (the American Genetic transformation Jo et al. 2014 variety Atlantic, the Dutch (cisgenics) variety Bintje and the Korean variety Potae9) Rpi-amr3i P. infestans S. americanum S. tuberosum Solynta Genetic transformation Witek et al. 2016a Research line 26 Rxo1 X. oryzae pv. oryzicola Zea mays Oryza sativa Genetic transformation Zhao et al. 2005 N TMV Tobacco Tomato Genetic transformation Whitham et al. 1996 Cf-9 Cladosporium fulvum Tomato Potato and tobacco Genetic transformation Hammond-Kosack et al. 1998 RB P. infestans S. bulbocastanum S. tuberosum Genetic transformation Bradeen et al. 2009 Rpi-vnt1.1 P. infestans S. venturii S. tuberosum Genetic transformation Foster et al. 2009 Bs2 Xanthomonas citri subsp. citri Capsicum chacoense Sweet orange (Citrus sinensis Genetic transformation Sendín et al. 2017 L. Osbeck) Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23  Table 2  (continued) Gene Pathogen/disease Donor Recipient Method References Pm3a × Pm3b, Pm3a × Blumeria graminis f. sp. tritici Wheat cv. Bobwhite Spring wheat cv. Bobwhite Genetic transformation Koller et al. 2018 Pm3d, Pm3b × Pm3d, and Pm3b × Pm3f Xa3 X. oryzae pv. oryzae indica rice var. Minghui 63 japonica rice varieties, Zhon- Genetic transformation Cao et al. 2007 ghua 11 and 02428 GmKR3 SMV, BCMV, WMV, and Glycine max (Soybean) Glycine max (Soybean) Genetic transformation (over- Xun et al. 2019 BPMV expression) Xa21, Xa5, and Xa13 Xanthomonas oryzae pv. Rice cv. ‘Jalmagna’ Breeding with MAS Pradhan et al. 2015 oryzae Xa21, xa13, and Pi54 X. oryzae pv. oryzae Rice var. JGL1798 Breeding with MAS Swathi et al. 2019 Xa21, xa13, Pi54 and Pi1 X. oryzae pv. oryzae Rice var. Tellahamsa Breeding with MAS Jamaloddin et al. 2020 Xa4, xa5, xa13, and Xa21 X. oryzae pv. oryzae Rice cv. Ciherang and Ranid- Breeding with MAS Biswas et al. 2021 han Pita + Pi3/5/i Magnaporthe oryzae Geng rice cultivars Breeding with MAS Gao et al. 2023 Pita + Pia M. oryzae Geng rice cultivars Breeding with MAS Gao et al. 2023 BCMV- Bean common mosaic virus; BPMV- Bean pod mottle virus; MAS- Marker-assisted screening; Rpi- Resistance to Phytophthora infestans; RPW8.1/8.2- Resistance to powdery mildew 8.1/8.2; Rvi6- Resistance to Venturia inaequalis 6; Rxo1- Resistance to Xanthomonas oryzae pv. oryzicola 1; SMV- Soybean mosaic virus; TMV- Tobacco mosaic virus; WMV- Watermelon mosaic virus Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23 13 11 12 Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23 technology have assisted in the successful introduction of Breeding for R gene‑mediated resistance resistance in many species, however, both suffer from their own set of drawbacks and limitations. In the recent past, the R genes have found essential roles in conventional resist- prospects of CRISPR/Cas (Clustered Regularly Interspaced ance breeding programs for decades and their potential has Short Palindromic Repeats/CRISPR-associated protein) been harnessed to develop disease-resistant cultivars by technology have been described for engineering improved introgression of the R gene(s) into previously susceptible resistance (Nagy et al. 2021). recipient lines through traditional breeding (Balconi et al. An increasing understanding of the molecular mecha- 2012). Traditionally, R genes were introduced one at a time nisms underlying R gene-mediated defense response along and maintained as crop monocultures. Monoculture crops with the discovery of novel R genes is providing the much- especially become susceptible to pathogens that become needed impetus to the development of newer resistant prevalent over time. On many occasions, complete resist- varieties. With the identification of novel R genes and the ance to one or more pathogenic strains may be provided by development of newer technologies, the future of resistance a single R gene. The loss of resistance in terms of specific- breeding and engineering in plants seems bright. Gain- ity, breadth of spectrum, and durability, however, arises as ing insights into the yet elusive molecular mechanisms of a major concern, as in many cases resistance imparted by a R gene-mediated resistance will prove to not only expand single R gene is successful only for the initial few years but the knowledge base for future use but will also promote the is later thwarted by newly emergent pathogen strains that utilization of R genes for the development of varieties with rapidly evolve to evade it by altering the corresponding Avr broad spectrum and more durable disease resistance. This gene with minor fitness penalty (McDowell and Woffenden review attempts to summarize in a comprehensive manner 2003; Dangl et al. 2013). One effective way that has been the R gene-mediated plant resistance and to collate new recommended to alleviate this problem of resistance dura- advances made towards the development of plants with bility is to reduce the selective pressure on pathogens by improved resistance to pathogens for the strategic manage- growing cultivars with diverse R genes (cultivar multilines) ment of diseases. or practicing rotation of multilines in the same field as suc- cessfully exemplified for controlling blackleg disease caused by Leptosphaeria maculans by rotating canola cultivars with different R genes (van de Wouw et al. 2018). Another R gene‑mediated disease resistance alternative strategy advocated for extended durability is the deployment of multiple R genes simultaneously in a phe- The introduction of resistance in plants has been an area of nomenon known as R gene pyramiding or stacking (Swathi great importance for enhancing agricultural benefits. It pre- et al. 2019; Jamaloddin et al. 2020; Biswas et al. 2021). R sents a valuable measure to mitigate the food scarcity caused gene pyramiding aims at combining multiple desirable R by various biotic agencies (Balconi et al. 2012; van Esse genes into a single genotype to improve disease resistance. et al. 2020). So far, breeding for the development of resistant Longer lasting durability of resistance is expected for such R varieties suitable for cultivation in environments with pre- genes pyramided in a species as the simultaneous accumula- vailing biotic pressures has been a desired method to control tion of mutations in more than one Avr gene by pathogenic various diseases and curtail crop losses. Identification of strains in order to escape detection is a low occurrence event, resistant wild relatives of crop plants and introgression of especially if cumulatively the mutations strongly impact vir- the resistance in cultivated crops has long been employed by ulence (Balconi et al. 2012; Dong and Ronald 2019). Gene plant breeders. More recently, with the advancement of bio- pyramiding using conventional breeding is performed by technology and the advent of plant genetic engineering tools, cross-breeding lines with preexisting R gene loci. However, it has been possible to incorporate R genes in susceptible it requires rigorous phenotyping for the selection of progeny lines in order to make them more resistant to the particular with desired R gene composition and thus as a remedy to pathogen(s). Many successful examples of the deployment this shortcoming, marker-assisted selection has surfaced as of these R genes have been elucidated during crop breeding a useful methodology. in addition to the biotechnological interventions for impart- Marker-assisted selection (MAS) uses DNA markers ing improved disease resistance against a range of pathogen to simplify the process of selection, particularly in cases systems, with still many new possibilities remaining to be where several R genes have been simultaneously introduced explored (Pandolfi et al. 2017; Dong and Ronald 2019; van or stacked into an elite genetic background (Das and Rao Wersch et al. 2020a; van Esse et al. 2020). Many of these 2015; Swathi et al. 2019; Jamaloddin et al. 2020; Rakosy- efforts have been concentrated on devising stable disease Tican 2020; Biswas et al. 2021; Rogozina et al. 2021). For control measures by imparting resistance that is not only example, cross-breeding followed by MAS has been used broad spectrum but also durable. to introgress three R genes (Xa21, Xa5, and Xa13) for 13 Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23 13 bacterial blight resistance in a deep-water rice cultivar ‘Jal- eliminate, thereby making the breeding economically unsus- magna’, with the resultant line with stacked R genes con- tainable (Balconi et al. 2012; Hasan et al. 2015; Lemaire ferring greater resistance to eight isolates of Xanthomonas et al. 2016). Advanced molecular biology approaches and oryzae pv. oryzae isolates (Pradhan et al. 2015). Similarly, transgenics technology have been shown to offer promising marker-assisted gene pyramiding has been used to introgress directions for the development of strategies to address these major resistance genes for bacterial blight and rice blast to problems. The issue of sexual incompatibility can be some- achieve various combinations, e.g., Xa21, xa13, and Pi54 in what alleviated by using the method of somatic hybridiza- rice variety JGL1798; Xa21, xa13, Pi54 and Pi1 in variety tion for introgression and stacking of R genes. With the use Tellahamsa and; Xa4, xa5, xa13, and Xa21 in rice cultivars of this method, it has been possible to transfer Rpi-blb1 and Ciherang and Ranidhan (Swathi et al. 2019; Jamaloddin et al. Rpi-blb3, two broad-spectrum resistance genes, along with 2020; Biswas et al. 2021; Pradhan et al. 2022). Geng rice two race-specific R genes (R3a and R3b), into the cultivated cultivars generated using combinations of Pita, Pi3/5/I, and potato from its sexually incompatible wild relative S. bulbo- Pia showed high resistance to rice blast, with Pita + Pi3/5/i castanum, which displays resistance to all known races of P. and Pita + Pia combinations yielding more stable pyramid- infestans (Rakosy-Tican et al. 2020). ing effects (Gao et al. 2023). Pyramiding of up to 5 blast- resistance genes has been achieved using marker-assisted Transgenic technology for R gene transfer backcrossing (MABC) and Kompetitive allele-specific PCR (KASP), whereby an additional blast-resistance gene, the Concerted efforts toward the identification and subsequent Pita-linked Pita2/Ptr gene was introgressed along with Piz, implementation of resistance-building strategies have paved Pib, Pita, and Pik in an elite rice cultivar (Zampieri et al. the way for the development of efficient approaches based 2023). Likewise, multiparental interspecific potato hybrids on advanced biotechnological methods. Some of these bio- with long-lasting late blight resistance have been developed technological interventions that have been verified for their that combine highly efficient Rpi (Resistance to P. infestans) utilization in conferring resistance by virtue of R gene intro- genes capable of recognizing multiple Avr genes (Rogozina duction or manipulation include by transgenic expression et al. 2021). MAS illustrated that these hybrids harbour at of R genes, R gene overexpression, induction of constitu- least 10 different Rpi genes with 4 to 6 Rpi genes in a single tive R gene, co-transformation mediated transgene stack- plant, which were initially identified in Solanum demissum, ing, mutagenesis-directed gene modification, and targeted S. bulbocastanum/S. stoloniferum and S. venturii (Rogozina genome editing (Balconi et al. 2012; Cesari 2018; Dong and et al. 2021). Likewise, lines with pyramided Rpp (Resistance Ronald 2019; van Esse et al. 2020; van Wersch et al. 2020a). to Phakopsora polysora) genes for resistance to Asian soy- Genetic transformation of plants for enhanced resistance has bean rust fungus P. pachyrhizi display improved resistance seen much progress due to the availability of cloned R genes than the ancestors containing single R genes (Meira et al. which, in turn, has allowed for the direct transfer of one or 2021; Panho et al. 2022). more R genes effective against a single pathogen species or With the use of markers, each R gene can be accurately race into elite lines within a single generation. The adoption traced through each breeding generation resulting in an of transgenic technology has also helped in overcoming the acceleration of breeding with more plant generations per barriers of sexual incompatibility and interspecies infertility year (van Wersch et al. 2020a). Although MAS has short- by eliminating the need for crossing. Furthermore, genetic ened the selection process to enhance the efficiency of gene modification of plants is able to surmount another major pyramiding inclusive of multiple loci using traditional constraint associated with breeding, i.e., linkage drags, as it breeding, it is expensive and still rather labour-intensive uses molecularly isolated and cloned R genes free from any and time-consuming (Cesari 2018; Dong and Ronald 2019). potentially negative aspect of linkage. These R genes can Identification of an appropriate R gene source is a funda- come even from different genetic backgrounds thus enabling mental step of resistance breeding in a given plant which the exploitation of a wider source of valuable R genes with- may be a wild type close relative or a more distantly related out any fitness penalty (Joshi and Nayak 2011). taxonomic group. Owing to this intrinsic requirement, breed- Transformation of susceptible lines with R genes cloned ing may not always be a feasible option due to associated from diverse sources has been reported to provide resistance problems, like sexual incompatibility between the recipi- to a range of pathogens (Pandolfi et al. 2017). One of the ent and the donor species and sterility of the interspecific/ first reports of R gene transfer using genetic transformation intergeneric hybrid which could render the whole breeding was demonstrated in tomato wherein a susceptible tomato program a failure (Balconi et al. 2012). Even if success- cultivar (Solanum lycopersicum L. cv. Moneymaker) was ful, resistance breeding may also bring about the introduc- transformed with the Pto gene, R gene related with resist- tion of undesirable traits (known as linkage drag) which, in ance to bacterial speck pathogen P. syringae, and was shown turn, may require backcrossing through many generations to to be devoid of typical symptoms and thus found resistant to 13 14 Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23 P. syringae pv. tomato strain (Martin et al. 1993). On simi- elements (Espinoza et al. 2013). The former uses a gene of lar lines, transgenic tomatoes carrying the tobacco N gene interest flanked by its own promoter and terminator, while and transgenic lines of potato and tobacco expressing the in the latter these regulatory elements may be self-derived tomato Cf-9 gene have been shown to exhibit HR in response or otherwise may be derived from a related but cross-com- to tobacco mosaic virus (TMV) and Cladosporium fulvum, patible species. Many prominent examples of successful R respectively (Whitham et al. 1996; Hammond-Kosack et al. gene pyramiding by genetic engineering exist for important 1998). Since these pioneering works, more such examples crops such as wheat, rice, and potato (Jo et al. 2014; Hae- of successful transformations with respect to disease resist- saert et al. 2015a; Kumari et al. 2017; Koller et al. 2018). R ance have accumulated (Pandolfi et al. 2017). Transgenic gene pyramiding for wheat Pm3 alleles that confer resistance potato lines expressing RB or Rpi-vnt1.1 from wild potato to powdery mildew (Blumeria graminis f. sp. tritici) has species S. bulbocastanum and S. venturii show enhanced been achieved via biolistic transformation in spring wheat resistance to late blight (Bradeen et al. 2009; Foster et al. cultivar Bobwhite that lacks endogenous Pm3 allele (Brun- 2009). Furthermore, maize R gene Rxo1, when expressed ner et al. 2011, 2012). Four such pyramided lines (Pm3a × in rice, imparts resistance to bacterial streak pathogen Xan- Pm3b, Pm3a × Pm3d, Pm3b × Pm3d, and Pm3b × Pm3f) thomonas oryzae pv. oryzicola, and tomato transgenic lines have been reported to enhance powdery mildew resistance expressing the pepper Bs2 R gene have been shown to be in field with no pleiotropic effects on plant development resistant to Xanthomonas sp. responsible for bacterial leaf and yield (Koller et al. 2018). Cisgenics has been efficiently spot (Zhao et al. 2005; Horvath et al. 2015). employed in the production of potato varieties resistant to The investigations on tomato Cf-9 and C. fulvum Avr9 late blight by transfer of multiple resistance genes from gene combination have also unveiled another strategy for related species, e.g., Rpi-sto1, Rpi-vnt1.1, and Rpi-blb3 from broad-spectrum resistance through co-expression of R and S. stoloniferum, S. venturii, and S. bulbocastanum, respec- Avr genes assembled in a single plant genotype by triggering tively, along with their innate regulatory sequences (Jo et al. HR and promoting acquired resistance (Hammond-Kosack 2014; Haesaert et al. 2015a). This method has also been suc- et al. 1998). However, taking into consideration the fact that cessful in developing scab-resistant cisgenic lines of apple the presence of both the effector and the NLR would lead through the transformation of M. domestica cv. Gala with to constitutive HR, which might result in plant death, this Rvi6 (Resistance to Venturia inaequalis, formerly HcrVf2) strategy will be beneficial only when genes are expressed gene against the ascomycete V. inaequalis from wild relative under artificially synthesized promoters designed specifi- Malus floribunda, including its own promoter and terminator cally to be pathogen inducible for tight regulation of their (Vanblaere et al. 2011, 2014). Co-transformation-mediated expression (Rushton et al. 2002). This has led to the con- transgene stacking of two major rice blast resistance R genes cept of pathogen inducible systems for enhanced resist- Pi54 and Pi54rh has enabled the widening of the resistance ance expressed under such designed promotors to avoid spectrum in a blast susceptible rice variety (japonica rice the negative effects of R gene overexpression, like stunted Taipei 309 or TP309) against different Magnaporthe oryzae growth and other agronomic traits, autoimmune phenotype, isolates (Kumari et al. 2017). Similarly, pyramiding of three and death (Mehrotra et al. 2011; Sendín et al. 2017). So far, Pi genes Pib, Pi25, and Pi54 in two rice varieties Kasalath only a few examples of successful testing of such induc- (indica variety) and Zhenghan 10 (japonica variety) has ible systems exist. For instance, the transfer of the Bs2 gene been shown to impart resistance to M. oryzae, however, it is from Capsicum chacoense when expressed under a patho- associated with pleiotropic effects in terms of yield penalties gen-inducible promoter in sweet orange has been described (Peng et al. 2021). to impart improved enhanced pathogen resistance and an Attempts have also been made to enhance disease resist- enhanced shoot survival in comparison to both the untrans- ance by R gene engineering by directed molecular evolu- formed and non-inducible controls (Sendín et al. 2017). tion. Hence, genetic engineering using targeted mutagenesis Gene pyramiding by co-transformation of multiple has emerged as an attractive method of engineering novel R genes has been shown to offer more durable resistance sources of disease resistance by modifying the known R against multiple diseases in a single step (Brunner et al. genes or manipulating related components of the defense 2011, 2012; Kumari et al. 2017; Rodriguez-Moreno et al. signaling, as well as the host proteins that are targets of 2017). Strategies like cisgenesis and intragenesis have been pathogen effectors (Cesari 2018; Dong and Ronald 2019). used for crop modification to improve disease resistance This may be achieved through random mutagenesis followed either through overexpression of preexisting R genes in a by mutation screens or targeted point mutations for directed species or by transferring R genes from a related species molecular evolution of NLRs and associated proteins for fab- (Espinoza et al. 2013; Holme et al. 2013). Both cisgenesis ricating new recognition motifs (Harris et al. 2013; Segretin and intragenesis also rely on genetic transformation but dif- et al. 2014; Stirnweis et al. 2014). Modification of the struc- fer in regards to the origin of promoter and other regulatory tural domain of R proteins can thus influence the disease 13 Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23 15 resistance spectrum, which may be exploited to develop RGA5, which when expressed in the heterologous host Nico- novel resistance or widen the spectrum of a resistance tiana benthamiana, is also able to perceive the AvrPikD, already in place. Various investigations have reported that without affecting its recognition specificity for its corre- modification of the recognition specificity and sensitivity of sponding effectors AvrPia and Avr1-CO39, resulting in the NLR proteins enables them to detect varied effector isoforms activation of defense. In a similar study, an RGA5-HMA2 and enhance their activation sensitivity. These modifications variant has been shown to recognize non-corresponding may involve the introduction of alterations in LRR and NB AvrPib from the M. oryzae which may be accredited to its domains in terms of changes in amino acid residues, both high structural similarity with the corresponding effectors of of which have been shown to widen the resistance spectrum RGA5, i.e., AvrPia and Avr1-CO39, although its mechanism of the NLR protein (Harris et al. 2013; Segretin et al. 2014; of triggering Pib-mediated resistance, which lacks an HMA Stirnweis et al. 2014). This has been verified for Rx protein domain, still remains elusive (Liu et al. 2021). In spite of its that mediates resistance against potato virus X (PVX), which conceivable utility, ID engineering is challenging as modi- when mutated in its LRR domain, is able to recognize a dis- fication of IDs may negatively influence the NLR function tantly related poplar mosaic virus (PopMV), but displays a by affecting the associations with other domains required for greater activation sensitivity when mutated in its NB domain effectors recognition (Cesari 2018). (Harris et al. 2013). The specificity of potato R3a, the NLR In an alternative strategy, the decoys enabling indirect responsible for resistance to P. infestans strains producing pathogen-derived effector recognition can be targeted for Avr3aKI effector, has been shown to become modified in engineering to broaden their recognition ability to accom- mutants with altered CC, NB, or LRR domains, thereby modate other effectors that employ a similar kind of modifi- enabling recognition of other Avr3a variants, including cation of the decoy protein (Kim et al. 2016). This approach Avr3aEM, that go undetected by the unmutated counter- of decoy engineering has been confirmed for PBS1, a part (Chapman et al. 2014; Segretin et al. 2014). In a sub- decoy target of a protease effector AvrPphB, as engineered sequent investigation, the same mutation when engineered forms of this decoy protein inserted with protease cleavage in the tomato ortholog I2 has been demonstrated to expand sequence from other protease effectors (i.e., AvrRpt2 of P. its spectrum for partial recognition of both P. infestans and syringae, NIa protease effectors of soybean mosaic virus Fusarium oxysporum (Giannakopoulou et al. 2015). Like- (SMV), tobacco etch virus (TEV) and turnip mosaic virus wise, mutations in the integrated sensor domain of the Pik (TuMV) in place of the original cleavage site are able to get group of NLRs, that recognize effectors of rice blast fungus cleaved by and thus detect respective pathogen-secreted pro- M. oryzae, produce new recognition motifs capable of rec- tease effectors and activate RPS5-dependent immunity (Kim ognizing other effectors of M. oryzae, while mutations in the et al. 2016; Helm et al. 2019). This highlighted the versatil- NB domain of Pm3, resistance protein against B. graminis f. ity of this approach which may be extended to other decoys sp. tritici resistance, fortifies its immune response (Stirnweis through modification of their ability to perceive other such et al. 2014; De la Concepcion et al. 2019). To further extend effector-induced post-translational modifications (PTMs), the resistance spectrum, a combination of both an increase and thus may be an attractive approach to provide resistance in specificity and sensitivity may be accomplished by com- to new pathogens (Kim et al. 2016; Kourelis et al. 2016; pounding modifications in both the LRR and NB domains Cesari 2018; Helm et al. 2019). (Cesari 2018). Another powerful method based on comparative genom- Engineering of integrated domains (IDs) has been ics has been developed and employed for the identification described as another promising approach for the creation of of genomic variants in R genes via selective capturing and novel recognition specificities in NLRs by way of introduc- sequencing, known as the RenSeq (Resistance gene enrich- ing targeted mutations into the ID or facilitating its replace- ment sequencing) (Jupe et al. 2013, 2014). With this method, ment, given prior knowledge exists regarding the molecular it will be possible to accelerate the cloning of novel R genes and structural basis of interaction of effector and the targeted which could be further applied to imparting disease toler- IDs of NLR receptors (Liu et al. 2021; Cesari et al. 2022). ance. The applicability of this method has been success- Rice NLRs RGA5 (Resistance Gene Analog 5) and Pikp-1 fully exemplified in Solanum americanum, a wild relative for recognition of distinct MAX (Magnaporthe Avrs and of potato, for the discovery of R gene Rpi-amr3i against P. ToxB-like) effectors, AvrPikD, AvrPia, and Avr1-CO39, infestans, whose transgenic expression in cultivated potato from M. oryzae rely on binding of effectors at their HMA imparts complete resistance to the pathogen (Witek et al. (Heavy Metal-Associated) IDs, albeit at different surfaces 2016a). Likewise, the method has been used for the iden- (Cesari et al. 2013; Maqbool et al. 2015). RGA5-HMA tification of late blight resistance gene Rpi-ver1 (for resist- variants fabricated with a high-affinity binding surface have ance to P. infestans) in Solanum verrucosum, another wild been developed via the introduction of residues of the Pikp-1 potato species, stem rust resistance genes Sr22 and Sr45 HMA domain for AvrPikD binding into the HMA domain of in mutagenized hexaploid bread wheat and Sr in Aegilops 13 16 Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23 tauschii ssp. strangulata, a wild progenitor of bread wheat Bs2 from tomato can function to recognize corresponding (Steuernagel et al. 2016; Chen et al. 2018; Arora et al. 2019). AvrBs2 and induce cell death in heterologous hosts from The technique has also been used for unraveling the genome- related species within the same family, like tobacco, potato and wide NLR complement for disease resistance in the wild pepper, but not in Arabidopsis, while the RPS2 (Arabidopsis grass Haynaldia villosa, a potential candidate for wheat Resistance to Pseudomonas syringae 2) fails to work in tomato improvement (Huang et al. 2022). (Tai et al. 1999; Hulbert et al. 2001). This might be attributed More recently, the emergence of genome editing technol- to a lack of the downstream signal transduction components ogies, especially the CRISPR/Cas technology, has opened in the recipient or otherwise be a reflection of the inability of new avenues for manipulating plant genomes by targeted transferred R protein to cooperate with available components editing for developing novel resistance in plant species as and networks in the heterologous system due to their diver- an effective strategy to control diseases (Barrangou and gence (Ellis et al. 2000). However, it is not a universal feature Doudna 2016; Dong and Ronald 2019). CRISPR/Cas may and, in some cases, transfer of certain R genes is possible even offer interesting prospects for future crop improvement between distantly related species (Xiao et al. 2003; Mendes CRISPR gene editing and may be used for targeted editing et al. 2010; Fradin et al. 2011; Afroz et al. 2011). For exam- of the LRR domain of NLRs to generate novel pathogen ple, the Arabidopsis R genes RPW8.1 (Resistance to Powdery recognition specificity for enhanced resistance (van Wersch Mildew 8.1) and RPW8.2 (Resistance to Powdery Mildew 8.2) et al. 2020a). A CRISPR-based method referred to as EvolvR maintain functionality in N. tabacum and N. benthamiana to has been developed to achieve such targeted mutagenesis provide broad-spectrum resistance to powdery mildew and and facilitate the directed evolution of existing alleles in the tomato Ve1 gene confers resistance against V. dahlia and order to produce novel alleles with altered ligand specific- V. alboatrum race 1 strains in transgenic A. thaliana. (Xiao ity, thereby conferring the ability to recognize a broader et al. 2003; Fradin et al. 2011). Another example of an effec- pathogen range (Halperin et al. 2018). In soybean, CRISPR/ tive interfamily transfer of the R gene is the rice Xa21 gene Cas9 technology has been used for promoting novel disease for resistance to X. oryzae pv. oryzae, the causal organism resistance via targeted chromosome cleavage of two tandem of rice bacterial blight, which has been reported to provide duplicated NLR gene clusters, Rpp1L (Resistance to Pha- enhanced resistance to citrus canker in oranges and to bacterial kopsora pachyrhizi 1 L.) and Rps1 (Resistance to P. sojae wilt in tomatoes caused by Xanthomonas axonopodis pv. citri 1), respectively (Nagy et al. 2021). The tandem arrangement and Pseudomonas solanacearum, respectively (Mendes et al. of R genes in the genome provides opportunities for recom- 2010; Afroz et al. 2011). binational rearrangements thus producing chimeric genes. Another setback has been proposed with regard to R gene CRISPR/Cas9-mediated chromosomal rearrangements in the pyramiding as concerns have been raised for the development two R gene clusters Rpp1L and Rps1 have been demonstrated of superpathogens by deployment of multiple R genes which to produce new resistance gene paralogs with intact open could cause the agriculture sector to bear overwhelming crop reading frames which are expected to confer new resistance losses (van Wersch et al. 2020a). Moreover, genome-editing specificities (Nagy et al. 2021). This method of artificially technologies may also raise alarm as in events of off-target generating chimeric paralogs by CRISPR/Cas9-mediated mutations, they are likely to wield deleterious effects on crops genome editing may be applicable to other R gene loci and (Cai et al. 2018). A consortium of various strategies has been may be used to improve plant resistance against diverse path- developed for minimizing the off-target effects of genome ogens. In an alternative strategy, disease resistance may be editing, however, its utilization is still subject to skepticism developed by inducing gain-of-function mutagenesis in the (Naeem et al. 2020). The most noteworthy drawback is the recessive r gene alleles to remove any nonsense mutations global public concern regarding the acceptance of genetically in the coding sequence by means of editing methods, such modified organisms (GMO), as well as the fear of the potential as prime editors, so as to allow them to act as pseudogenes introduction of engineered genes into wild populations. Con- (Molla and Yang 2019). sequently, strict regulatory norms, have been established for Although transgenic technology with respect to R gene approval of the use of both genetically-modified and genome- transfer has seen much progress over recent years for potential edited organisms in several countries, thereby restricting the use in generating resistant plants, it is predisposed to multiple use of GM crops (Bawa and Anilakumar 2013; Lassoued et al. shortcomings. Firstly, R gene introgression, in general, has 2019). been more successful in conferring resistance when the donor and recipient species are phylogenetically related (Tai et al. 1999; Hammond-Kosack and Parker 2003). This deficiency is termed as ‘restricted taxonomic functionality’ (RTF) whereby R gene transfer becomes fruitless as the transgene is not effec- tive in the heterologous host (Tai et al. 1999). For example, 13 Journal of Plant Biochemistry and Biotechnology (January–March 2024) 33(1):5–23 17 Conclusions and perspective insights in plant–pathogen interactions using molecular and omics approaches. The knowledge so generated will Sustainable crop production is one of the most significant have to be evaluated for applicability in crop improvement yet very challenging target as pathogens continue to evolve for disease tolerance and for realizing the full potential of under selection pressure. The cultivation of resistant crop the available R gene pool in a way forward for the effective varieties is the most effective method of disease control. management of diseases. A great deal of effort has been dedicated to the develop- ment and implementation of approaches for making crops Author contributions All authors contributed to the conception and more resistant to diverse pathogens so as to reduce crop design. The information collection was done by Aditi Tailor who also losses incurred annually. With the goal of deciphering new prepared the first draft of the manuscript. Critical review of this draft opportunities for crop improvement for broader and more was done by Satish C. Bhatla and his suggestions lead to further refine- durable pathogen resistance, there is a need to expedite ment of the draft, thus bringing it to its present state. the identification and utilization of genetic determinants Funding Authors also declare that no funding was received to assist conferring broad-spectrum resistance without any yield with the preparation of this manuscript. penalties. During the last three decades, many valuable discoveries have been made regarding the identity of R Declarations proteins and their corresponding effectors, modes of effec- Conflict of interest The authors declare that they have no conflict of tor recognition as well as the R gene-mediated immune interest. signaling pathways and related components, but a lot more remains to be elucidated. For many important plant spe- cies, limited resistance resources have been identified so References far that are available for use. Only a small subset of resist- ance genes has been cloned which, in turn, is restricted Adachi H, Derevnina L, Kamoun S (2019) NLR singletons, pairs, and to some major diseases, such as wheat rust, rice blast and networks: evolution, assembly, and regulation of the intracel- blight, and a large majority of resistance genes are yet lular immunoreceptor circuitry of plants. 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