Understanding the Plant-microbe Interactions in CRISPR/CAS9 Era: Indeed a Sprinting Start in Marathon.

Plant-microbe interactions can be either beneficial or harmful depending on the nature of the interaction. Multifaceted benefits of plant-associated microbes in crops are well documented. Specifically, the management of plant diseases using beneficial microbes is considered to be eco-friendly and the best alternative for sustainable agriculture. Diseases caused by various phytopathogens are responsible for a significant reduction in crop yield and cause substantial economic losses globally. In an ecosystem, there is always an equally daunting challenge for the establishment of disease and development of resistance by pathogens and plants, respectively. In particular, comprehending the complete view of the complex biological systems of plant-pathogen interactions, co-evolution and plant growth promotions (PGP) at both genetic and molecular levels requires novel approaches to decipher the function of genes involved in their interaction. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) is a fast, emerging, precise, eco-friendly and efficient tool to address the challenges in agriculture and decipher plant-microbe interaction in crops. Nowadays, the CRISPR/CAS9 approach is receiving major attention in the field of functional genomics and crop improvement. Consequently, the present review updates the prevailing knowledge in the deployment of CRISPR/CAS9 techniques to understand plant-microbe interactions, genes edited for the development of fungal, bacterial and viral disease resistance, to elucidate the nodulation processes, plant growth promotion, and future implications in agriculture. Further, CRISPR/CAS9 would be a new tool for the management of plant diseases and increasing productivity for climate resilience farming.

Introduction

Nature has multiple millions of microbes, which are continuously evolving and simultaneously interacting among themselves as well as with plants, animals, and the envclass="Gene">ironment. In class="Chemical">plants, microbes reside in the rhizosclass="Chemical">phere and additionally class="Chemical">present as endoclass="Chemical">phytes in roots and shoots. Based on the nature of the interaction, class="Chemical">plant-microbe interactions can be broadly categorized into beneficial and harmful. The class="Chemical">plant-beneficial (class="Chemical">pan class="Chemical">PB) microbes produce phyto-hormones, which support plant growth and afford protection against various plant pathogens [1]. As a direct mechanism, PB microbes enhance plant growth through biological nitrogen fixation, phosphorous uptake and production of phytohormones specifically, indole-3-acetic acid (IAA), gibberellic acid (GA) and cytokinins [2-4]. As an indirect mechanism, PB microbes suppress plant pathogenic microbes by producing different antibiotics, and promote induced systemic resistance in plants [5-8]. In contrast, many plant pathogenic (PP) microorganisms cause devastating diseases in various crops. Plant diseases extensively reduce crop yield and are considered as one of the major threats to food security worldwide [9]. Diseases caused by pathogens are generally controlled by the application of pesticides. Though pesticides play a significant role in sustainable food production and food security, their negative environmental impact and pesticide resistance are few major concerns in its usage [10]. The development of inherent genetic resistance to diseases is one of the sustainable approaches for ensuring food security. Several resistance genes (R genes) have been identified and successfully utilized in marker-assisted backcross breeding for the development of tolerant varieties in multiple crops [11]. However, efforts are persistent in the development of durable resistance to diseases. Recently, a genome editing technology named as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) is becoming highly prominent for understanding the biological function of genes in plants. Therefore, exploring the molecular components of plant-pathogen interactions using CRISPR-Cas9 based genome editing tools will be remarkably useful in developing durable, eco-friendly and sustainable disease management strategies.

Plant-microbes interaction in CRISPR/ CAS9 era

The interaction between plants and microbes can be either synergistic or antagonistic and elicit multiple responses. class="Chemical">Pathogens inclass="Chemical">pan class="Chemical">fect plants and initiate the process of colonization, utilization of plant nutrition, which results in disease symptoms. In return, plants activate a defense response through antimicrobial and stress response pathways. The identification, isolation, and characterization of multiple resistance genes in plants were made possible adopting different techniques/methods inclusive of map-based cloning [12], whole-genome sequencing through next-generation sequencing (NGS) technologies [13], comparative genomics [14], RNA-seq analysis [15, 16], RNA-interference (RNAi) [17], proteomics [18, 19] and availability of mutant resources of genes used for functional characterization [20]. These technologies greatly assisted in translating the findings for application in the crop improvement programs [21]. For instance, reduction in the cost of sequencing has led to the sequencing of several plant genomes, including lupin (Lupinus angustifolius), a leguminous plant sequenced with the primary objective of understanding plant-microbe interactions [22].

CRISPR/CAS9 technique

CRIclass="Chemical">PSR-class="Chemical">pan class="Gene">Cas9 is a relatively new technique, which is being increasingly used nowadays for functional genomics [23]. Moreover, CRISPR/CAS9 is an emerging, fast, precise, eco-friendly and efficient tool to address the challenges in agriculture, especially deciphering plant-microbe interaction for resistance development in crops. Additionally, CRISPR-Cas9 is more robust and specific in gene knockdown when compared to RNAi, which induces partial gene silencing [24]. Historically, a repetitive sequence of 21-40bp was identified in E. coli, which was instrumental in the development of gene-editing technology. Later, CAS proteins (CRISPR associated proteins) and repetitive sequences were predicted to be involved in prokaryotic immunity [25]. Specifically, gRNA (guideRNA) drives the CAS proteins to the complementary DNA sequence and induces DNA breaks. Further, first-generation CRISPR gene-editing technology has been successfully utilized to induce site-specific double-stranded breaks in the genome, exploiting two catalytic nuclease domains, namely HNH and RuvC, each cleaving a strand of DNA [26]. Site-specific cleavage by CAS9 is facilitated through single guide RNAs (sgRNAs) [27]. The second-generation CRISPR editing tool is capable of precisely converting a single base into another (Cytosine (C) to Thymine (T)) without causing double-strand DNA breaks [28, 29]. Moreover, with the improvement in ribosome engineering techniques, editing accuracy of Cas9 endonuclease has been enhanced by engineering hairpin secondary structures in guide RNA that brought about the concept of tuning CRISPR-Cas9, which increased specificity by several orders of magnitude [30].

CRISPR/CAS9 mediated resistance to plant diseases

Disease management using CRISclass="Chemical">PR/class="Chemical">pan class="Gene">CAS9 is considered an emerging tool for plant disease management [31]. Besides, CRISPR/CAS9 has also been used for genetic manipulation of microorganisms for enhancing microbial benefits toward crops [32]. Recently, the CRISPR-Cas9 system was developed in order to decipher the mechanisms of Bacillus-plant interactions [33]. Therefore, the present review provides an overview of the application of CRISPR/CAS9 in plant-microbe interactions, particularly for the development of disease resistance and improvement of growth promotion, biocontrol ability of beneficial microbes.

CRISPR/CAS9 and resistance to fungal diseases

class="Disease">Plant diseases caused by funclass="Chemical">pan class="Chemical">gal pathogens are considered to be the dominant factors responsible for the detrimental effect on plant yield. Nowadays, the emergence of virulent strains due to climatic changes is posing a far greater threat to crop production [34]. Besides, 30% of emerging diseases are caused by fungal pathogens, which can easily overcome R gene-mediated resistance due to high genetic diversity and evolutionary flexibility [35]. Therefore, gene-specific editing using the CRISPR/CAS9 approach will greatly assist in the development of durable resistance to fungal diseases. Rice blast caused by Magnaporthe oryzae is considered the most devastating disease worldwide and ranked one of the top fungal pathogens affecting food crops [36]. Previous studies reported that knockdown of ethylene-responsive factors (OsERF922) using RNAi approach enhanced disease resistance [37, 38]. In support of these findings, CRISPR/CAS9 based targeting of the OsERF922 gene in rice reduced blast lesion-symptom as compared to wild type in seedling and tillering stages. Besides, a significant difference in the agronomic traits was not observed between mutants and wild types. However, evaluation of the percent reduction in disease symptoms using multiple isolates within a geographical region will determine the efficacy of edited lines for commercial cultivation. In addition, ERF922 gene expression was up-regulated not only during infection of M. oryzae but also in abiotic stresses. Further, editing of the other three blast inducible ERF genes (OsBIERF1, OsBIERF3 and OsBIERF4) may provide additional insights into the role of ethylene response factors in regulating blast resistance in rice [39]. Similarly, the editing of a well-characterized Pi21 gene also enhanced resistance to blast fungus in rice [40]. Moreover, RNA-seq analysis between Pi21 and its loss-of-function lines showed pathogen-associated molecular patterns as one of the mechanisms conferring durable resistance [41]. Therefore, yeast two-hybrid assay of Pi21 protein with effector proteins will decipher the upstream signaling components in the Pi21-M.grisea interaction. Further, gene manipulation of exocyst subunit OsSEC3A and OsMPK5 via CRISPR/CAS9 enhanced the resistance against M. oryzae. However, both the mutants of sec3a and mpk5 had showed dwarf phenotype and susceptibility to abiotic stresses, respectively [42-44]. Specifically, localization studies on the protein translocation dynamics of SEC3A: GFP into the plasma membrane (PM) during pathogen interaction and its differential loading into PM might provide us vital clues to decouple the enhanced resistance and plant development traits. Similarly, MPK5 protein interaction network and downstream regulators responsible for providing disease resistance require further investigation. Therefore, the above-mentioned examples of Sec3a and MPK5 suggest that knockdown might result in disease trade-off and different editing strategy is required for genes having multiple roles in plants.

class="Species">Downy mildew and class="Chemical">powdery mildew are the most devastating class="Chemical">pan class="Disease">fungal diseases encountered in grape cultivation. A mildew locus O (MLO) gene up-regulated during Erysiphe necator infection induces susceptibility. RNAi based approach for silencing MLO S-genes viz., VvMLO7, VvMLO6 and VvMLO11 drastically reduced the severity (77%) of powdery mildew disease. Additionally, resistance to powdery mildew disease in other crops was provided by the silencing of MLO S genes [45, 46]. Three MLO alleles with TALEN-induced mutations were also conferred resistance to powdery mildew fungus Blumeria graminis f.sp. tritici in bread wheat [46, 47]. Furthermore, MLO-7 gene lines edited through CRISPR-Cas9 have been developed for enhancing resistance to downy mildew in grapes [48]. Mildew locus (MLO-S) genes form distinct clades and are evolutionarily conserved transmembrane domain proteins, which negatively regulate the penetration of powdery mildew fungus. Therefore, orthologous editing of MLO-S genes could be a prominent strategy in the development of mildew resistance in diverse crops, including fruits and vegetables [49, 50]. Besides, cell wall thickening was identified as one of the probable mechanisms contributing to resistance in the loss-of-function of the MLO1 gene in cucumber [51]. Recently, a medley of regulators were identified in regulating the cell wall thickening in plants [52]. Thus, enquiring the role of MLO signaling in regulating the medley of regulators, especially MYB and NAC genes involved in cell wall thickening, would be an attractive research hypothesis in exploring the mechanistic understanding of powdery mildew resistance. Similarly, CRISPR based knock-down of downy mildew resistance 6 (DMR6) also conferred resistance to downy mildew disease in grapevine. Thus, multiplex editing of MLO and DMR6 genes in grapes could provide durable resistance to downy mildew in grapes. Interestingly, editing of the tomato SlDmr6-1 gene also conferred resistance to different pathogens viz., Phytophthora capsici, Pseudomonas syringae, and Xanthomonas species [53]. Since dmr6 gene increased salicyclic acid levels, thus targeting the orthologs in monocots and dicots might provide additional information on the development of broad-spectrum resistance and its associated pleiotropic effects. However, a slight reduction in plant height and its associated yield penalty due to the editing of the DMR6 gene needs to be better understood, considering its beneficial effects of plant immunity.

Ascomycetous fungus class="Species">Leptosphaeria maculans causing ‘blackleg’ disease in oilseed croclass="Chemical">ps such as class="Chemical">pan class="Species">Brassica napus is a major problem worldwide. Besides, only limited information is available regarding genes involved in conferring resistance against L. maculans. Gene knock-down through the CRISPR-Cas9 approach in L. maculans identified a gene cluster responsible for the synthesis of pathogenicity factor, abscisic acid (ABA), which increases pycnidiospore germination and appressorium formation [54, 55]. In addition, ABA has been reported to restrict the growth of some beneficial fungi like Aspergillus nidulans, suggesting that CRISPR-Cas9 can be used to unravel complex microbiome interactions [56]. Therefore, deregulating the components of ABA perception in plants during L. maculans infection might be considered a double-edged sword due to its effects on pathogen restriction and biocontrol agent promotion. Phytophthora spp is disease-causing agent of Theobroma cacao and reduces its yield drastically. A gene named Non-expressor of Pathogenesis-Related 1 (TcNPR1) acts as a major regulator of the defense system in cocoa [57]. Moreover, Arabidopsis NPR3 gene negatively regulates NPR1 activity [58]. Further, knock-down of TcNPR3 transcripts in cocoa leaf tissues demonstrated enhanced resistance to P. tropicalis infection. Thus, the CRISPR-Cas9 approach could effectively complement the knock-down of NPR3 transcripts for resistance development in cocoa. One of the important factors influencing the efficiency of CRISPR/CAS9 systems is the generation of homozygous mutations in the first generation, effective targets editing and elevated expression of Cas9 protein [59]. This is vital for plants having long generation time, particularly woody plants such as grape. In a report, Wang et al., (2018) [60] investigated the efficiency of CRISPR/CAS9 mediated targeted mutagenesis in the first generation of grape mutants. Results demonstrated that knockout of the VvWRKY52 gene increased disease resistance against Botrytis cinerea infection as compared to wild type plants. Further, there was no significant difference in phenotype between wild-type and biallelic edited plants and concluded that CRISPR/CAS9 can be efficiently utilized for the specific genome editing in the first generation plants. Moreover, efficient disease control found in edited lines having early termination of translation indicates efficient target site selection for obtaining better disease response. Most of the loss-of-function tools result in hemizygous/heterozygous mutants as compared to CRISPR-Cas9. Thus, homozygous mutants generated through CRISPR-Cas9 will be remarkably useful in woody crops for early ascertainment of phenotypic response. The list of genes edited through CRISPR/CAS9 related to the development of resistance is given in Table (.

Table 1

List of target genes edited through CRISPR/CAS9 in different crops for understanding plant-microbe interaction and disease resistance.

S. No.	Crop	Targeted Pathogen					Target Genes						Signaling Pathway/Processes				Physiological Function/Disease Response	References
Bacterial Diseases
1.	Citrus sinensis(Citrus)	Xanthomonascitri sub sp. citri (Xcc)and Xanthomonasaxonopodis					LOB1						TALE effectors binding				Susceptibility factor against Xcc	[64, 123]
		Xanthomonascitri sub sp. citri (Xcc)					WRKY22						Salicylic acid (SA) regulated defense				Induces pathogen-triggeredImmunity	[124]
2.	Malus domestica(Apple)	Erwinia amylora					DIPM-1, DIPM-2, DIPM-4						Leucine-rich repeat receptor kinase				Reduces host susceptibility	[48]
3.	Oryza sativa(Rice)	Xanthomonas oryzaepv. oryzae					OsSWEET11,13,14						Sucrose efflux transporter				Non-availability of sugar metabolites and broad-spectrum resistance	[73, 125]
4.	Solanum lycopersicum (Tomato)	Pseudomonas syringae, Xanthomonas spp.					DMR6-1, DMR6-2						Inactivation of salicylic acid to 2,3-dihydroxybenzoic acid				Broad-spectrum resistance	[53]
		Pseudomonassyringae pv. Tomato					SlJAZ2						Jasmonic acid-mediated regulation of stomata				Stomatal opening and disease susceptibility	[76]
Fungal Diseases
S. No.	Crop	Targeted Disease					Target Genes						Signaling Pathway/Processes				Physiological Function/Disease Response	References
1.	Gossypium hirsutum(Cotton)	Verticillium dahlia					GhMYB25						Transcription regulation for fiber and trichome development				Enhanced resistance toWilt	[126]
2.	Oryza sativa(Rice)	Magnaporthe oryzae					OsSEC3A						Enhanced salicylic acid signaling				Disease resistance and plant development	[42]
							OsERF922						Ethylene mediated signaling				Reduced susceptibility to blast disease	[38]
							Pi21						Induces pathogen-associated molecular pattern response				Durable resistance	[40]
		Magnaporthe grisea and Burkholderia glumae					OsMPK5						MAP kinase pathway				Enhanced resistance	[43]
3.	Solanum lycopersicum (Tomato)	Phytophthora capsici					SiDMR6-1, SiDMR6-2						Inactivation of salicylic acid to 2,3-dihydroxybenzoic acid				Broad-spectrum resistance	[53]
		Oidium neolycopersici					SlMlo1						Negative regulation of vesicle-associated and actin-dependent defense pathways				Broad-spectrum and durable resistance	[127]
		Fusarium oxysporum f.sp. lycopersici					Solyc08g075770 (CYCLOPS)						Inhibition of mycorrhizal colony				Enhanced defense response	[128]
S. No.	Crop		Targeted Disease						Target Genes		Signaling Pathway/Processes				Physiological Function/Disease Response			References
4.	Theobroma cacao(Cacao)		Phythopthora tropicalis						NPR3		Suppressor of defense response				Enhanced disease resistance			[129]
5.	Triticum aestivum(Wheat)		Blumeria graminis f.sp. tritici						TaMLO		Negative regulation of vesicle-associated and actin-dependent defense pathways				Broad-spectrum and durable resistance			[47]
6.			Fusarium graminearum						TaLpx-1		Lipoxygenase activity				Enhanced resistance response			[130]
			Blumeria graminis f.sp. tritici						TaEDR1		MAP kinase pathway				Resistance response			[47]
7.	Vitis vinifera (Grape)		Botrytis cinerea						WRKY52		Transcription regulation of pathogenesis-related genes				Transcriptional reprogramming to regulate disease resistance			[60]
			Erysiphe necator						VvMlo7		Negative regulation of vesicle-associated and actin-dependent defense pathways				Broad-spectrum and durable resistance			[52]
8.	Brassica napus		Sclerotiania						WRKY11, WRKY17		Suppressor of jasmonic acid and salicylic acid signaling				Enhanced resistance			[131, 132]
Viral Diseases
S. No.	Crop		Targeted Pathogen						Target Genes		Signaling Pathway/Processes				Physiological Function/Disease Response			References
1.	Nicotiana benthamiana		Beet severe curly top virus (BSCTV)						A7, B7 and C3 sites		Replication				Interferes with replication of virus			[79]
			TYLCV, BCTV, MeMV,CLCuKoV*						CP, Rep, IR		Virus maturation				Interferes with replication of virus			[79, 80]
			Cotton Leaf Curl Multan virus (CLCuMuV)						IR		Transcriptional regulation				Inhibits bidirectional transcription			[133]
2.	Nicotiana benthamiana,Arabidopsis thaliana		Bean yellow dwarf virus (BeYDV)						LIR, Rep/RepA		Replication				Interferes with replication of virus			[80]
			Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV)						Sequence target sites		Replication				Cleavage of RNA viruses			[85]
3.	Arabidopsis thaliana		Turnip mosaic virus(TuMV)						eIF(iso)4E		Initiation factor				Disruption of viral translation			[84]
			Cauliflower mosaicvirus (CaMV)						CP		Virus assembly				Disables the assembly of virus			[134]
4.	Oryza sativa L.Japonica		Rice tungro sphericalvirus (RTSV)						eIF4G		Initiation factor				Disruption of viral translation			[86]
5.	Tomata		Tomato yellow leaf curl virus (TYLCV)						CP		Virus assembly				Disables the assembly of virus			[132]
6.	Hordeum vulgare		Wheat dwarf virus (WDV)						MP/CP, Rep/RepA, LIR		Replication				Interferes with replication of virus			[135]
7.	Musa spp. (banana)		Endogenous banana streak virus (eBSV)						ORF1, ORF2, ORF3		Strand cleavage				Knockout of dsDNA			[136]
S. No.	Crop				Targeted Pathogen		Target Genes					Signaling Pathway/Processes				Physiological Function/Disease Response		References
8.	Manihot esculenta				African cassava mosaic virus (ACMV)		AC2, AC3					Replication				Virus resistance		[137]
					Cassava brown streak virus(CBSV) and Ugandan cassava brown streak virus (UCBSV)		Novel cap-binding proteins (nCBP-1), and nCBP-2)					Translation and virus movement				Interferes with translation and movement of the virus		[138]
9.	Cucumis sativus				CVYV, ZYMV, PRSV-W**		eIF4E					Initiation factor				Broad virus resistance		[139]
10.	Solanum tuberosum				Potato virus Y (PVY)		Coilin gene					Host interaction				Impairment of host and virus interaction		[140]
Beneficial Microbes
S. No.	Crop				Targeted Trait		Target Genes					Signaling Pathway/Processes				Physiological Function		References
1.	Lotus japonicas				Biological N fixation		LjLb1, LjLb2, LjLb3					Oxygen binding				White color non-functionalnodules		[103]
2.	Vigna unguiculata				Biological N fixation		RLKs					Receptor-like kinase signalling				Absence of nodules		[104]
3.	Glycine max				Biological N fixation		Rfg1					Strain-specific response				Symbiosis specificity		[105]
4.	Parasponia andersonii				Biological N fixation		PanHK4, PanEIN2,PanNSP1, PanNSP2					Cytokinin, ethylene pathway				Conservation of nodulationin trees		[107]
5.	Medicago truncatula				Biological N fixation		MtGA2Ox10					Gibberellin metabolism and signaling				Suppressed the infectionthread formation		[109]
							NFS2					Biogenesis of Fe-S cluster				Strain specificity		[103]
Phytopathogens
S. No.	Pathogen					Targeted Disease	Target Genes					Signaling Pathway/Processes				Physiological Function/Phenotype		References
1.	Alternaria alternate					Blight	pyrG (orotidine-5-phosphate decarboxylase gene)					Regulation of pyrimidine biosynthesis				Auxotrops for uracil anduridine		[110]
							pksA(polyketide synthase)					Melanin biosynthesis pathway				Disruption results in white colonies		[110]
2.	Colletotrichumsansevieriae					Anthracnose	SCD1					Biosynthesis of dihydroxynaphthalene (DHN)-melanin				Lacks melaninbiosynthesis resulting in white colonies		[113]
3.	Fusariumoxysporum					Wilt	BIK1 polyketide synthase					Synthesis of bikaverin				Lacks bikaverin synthesis		[111]
							URA5					Uracil biosynthesis				Resistance to 5-fluoro-orotic acid and hygromycin		[111]
S. No.	Pathogen			Targeted Disease				Target Genes		Signaling Pathway/Processes				Physiological Function/Phenotype				References
4.	Fusarium proliferatum			Vascular wilt/Root Rot				FUM1		Biosynthesis of fumonisins B1				Absence of fumonisins and resistant to hygromycin B				[115]
5.	Phytophthora capsici and P. sojae			Phytophthora blights				PcORP1		Sporangia synthesis				Reduction in sporangia formation				[112]
6.	Phytophthora sojae			Damping-off				Avr4/6		Effector protein				Avirulency				[112]
7.	Sclerotinia sclerotiorum			Stem rot/ White mold				Ssoah1		Sclerotia formation				Reduced sclerotium				[114]
								Sspks13		Melanin biosynthesis				Absence of pigmentation				[114]

*Tomato yellow leaf curl virus (TYLCV), Beet curly top virus (BCTV), Merremia mosaic virus (MeMV), Cotton leaf curl, Kokhran virus (CLCuKoV) **Cucumber vein yellowing virus (CVYV), Zucchini yellow mosaic virus, (ZYMV), Papaya ringspot virus (PRSV-W).

CRISPR/CAS9 and resistance to bacterial diseases

class="Chemical">Plant bacterial class="Chemical">pathogens can sclass="Chemical">pread raclass="Chemical">pidly and establish eclass="Chemical">pidemics in a short term class="Chemical">period. Esclass="Chemical">pecially, the management of bacterial class="Chemical">pathogens is class="Chemical">profoundly difficlass="Chemical">pan class="Chemical">cult because of their accelerated multiplication and high diversity. The characterized resistant (R) or susceptibility (S) genes are the major targets in marker-assisted breeding for the development of bacterial disease resistance [61]. Apart from traditional map-based cloning and transgenic methods, CRISPR/CAS9 tool has also been used to achieve resistance to bacterial diseases in various crops. Citrus canker is a severe disease in citrus caused by Xanthomonas citri subsp. citri (Xcc), resulting in huge economic losses worldwide [62]. Hu et al. (2014) [63] demonstrated that citrus Lateral Organ Boundry 1 (CsLOB1) gene is responsible for disease-susceptibility in bacterial canker disease. During Xcc

class="Disease">infection, class="Chemical">pan class="Species">Xcc-derived transcription activator-like effector (TALE), i.e PthA4 is translocated into plant cells and induced CsLOB1 expression resulting in canker development. Specifically, Xcc-derived effector protein PthA4, specifically binds to effector binding elements (EBEPthA4) in the CsLOB1 promoter region (EBEPthA4-CsLOBP) to activate its gene expression. Thus, promoter editing of the EBEPthA4 cis-regulatory element of CsLOB1 showed resistance to bacterial canker disease in Duncan grapefruit. Similarly, the coding region of CsLOB1 was targeted via CRISPR/CAS9 in Wanjincheng Orange [64] and provided enhanced resistance to Xcc signifying the importance of targeting LOB1 for the development of canker resistance in plants. LOB genes are essential for pattern formation in meristem cells [65], and few genes showed differential expression during biotic stress [66]. Moreover, CsLOB1 gene expression was correlated with hypertrophy and hyperplasia, and few downstream genes have also been identified to be direct targets of CsLOB1 [67]. The co-expression of LOB1 and its downstream genes in relation to pathogen colonization could provide mechanistic insights for the induction of hypertrophy and hyperplasia. Thus, LOB homologs could be potential targets for resistance development in crops.

Another best example is the control of class="Disease">bacterial leaf blight (BB) disease in class="Chemical">pan class="Species">rice caused by Xanthomonas oryzae pv. oryzae (Xoo). In rice, BB is one of the major pathogens and causes yield losses of up to 50% and sometimes even complete yield loss [68]. The Xoo pathogen through the type III secretion system translocates the transcription activator-like effector (TALE) proteins into host cells and induces the expression of susceptibility factor genes (S genes) in plants [69-72]. Xoo produces PthXo2 (effector protein) that induces OsSWEET13 (S gene) expression and results in bacterial blight disease. Mutagenesis in the OsSWEET13 coding region via the CRISPR/ Cas9 system provided enhanced resistance to Xoo infection. Similarly, AvrXa7 is another TALE protein, which targets OsSWEET14 in rice. Gene editing in the OsSWEET14 promoter prevented the binding of AvrXa7 and consequently resulted in disease resistance. Recently, Oliva et al. (2019) [73] demonstrated that mutations in SWEET gene promoters of SWEET11, SWEET13 and SWEET14 provided a robust, broad-spectrum resistance to Xoo pathogen in rice. Thus, cis-TALE CRISPR (cis represents the promoter binding region of TALE effector) is an efficient disease control strategy in plants, which needs to be exploited in multiple crops. Further, differential disease response of multiple Xoo strains in indica and japonica varieties reinforces the requirement of evaluation of multiple pathotypes for disease screening. Additionally, SWEET11 and SWEET14 have also been found to be essential for reproductive development [74]. Thus, even though Oliva et al. (2019) [73] work showed no effect on percent spikelet fertility, three genes edited lines need to be evaluated under water-limiting or nutrient-limiting conditions for understandings its reproductive fitness. In tomato, Pseudomonas syringae pv. tomato (Pto) is the causal organism of bacterial speck disease. A refined strategy to manipulate hormonal crosstalk has been evolved in Pto strain DC3000 through the synthesis of coronatine (COR), which mimics the bioactive JA hormone. In plants, JA isoleucine (JA-Ile) stimulates stomatal opening, which facilitates bacterial invasion and promotes leaf colonization [75, 76]. Ortigosa et al. (2019) [77] showed SlJAZ2 is a major co-receptor of COR in stomatal guard cells of tomato. Further, the editing of the SlJAZ2 gene prevented stomatal reopening induced by COR and conferred resistance to Pto DC3000. Additionally, neither water use nor resistance to necrotrophic pathogen was affected. Moreover, salicyclic acid regulated factors involved in imparting resistance in addition to stomatal closure needs to be understood independently for trade-off effects. This novel strategy provides insights into the trade-off between biotrophs and necrotrophs in plants. Therefore, targeted knockout of susceptibility genes or/and negative regulators through genome editing provides a powerful strategy for disease resistance breeding.

CRISPR/CAS9 and resistance to plant viruses

class="Chemical">Plant vclass="Chemical">pan class="Gene">iruses are emerging as a major challenge in agriculture and horticulture worldwide. Viruses are obligate parasites, which depend on host cells for their survival and replication. Chemical control is specifically unavailable for the management of viral pathogens. Therefore, the development of genetic resistance to viruses is a significant alternate strategy for the control of viral diseases. RNAi is successfully employed in the control of viral diseases. Recently, the CRISPR/CAS9 system has been utilized to impart virus resistance by targeting either the viral genome or susceptibility genes of hosts [78]. Two families of ssDNA viruses (Geminiviridae and Nanoviridae) are known to infect plants. Geminiviridae is the largest known family of single-stranded DNA viruses, which infect both dicot and monocot plants, causing extensive crop losses globally [79]. Most of the CRISPR/CAS9 mediated viral resistance has been demonstrated against geminiviruses by targeting ssDNA of mono and bi-partite genome containing coat protein (CP), replication (Rep), intergenic region (IR) and nanonucleotide sequences [80] (Table ). CRISPR/CAS9 system imparting resistance to geminiviruses has been demonstrated in Beet severe curly top virus (BSCTV) and Bean yellow dwarf virus (BeYDV) in Arabidopsis thaliana and Nicotiana benthamiana, respectively. The plants expressing viral gene-specific sgRNA-Cas9 exhibited lower viral titre or delayed accumulation of viruses and reduced symptom expression. Similarly, Ali et al. (2015) [81] reported the efficiency of editing various coding and non-coding regions such as viral coat protein (CP), replication protein (Rep) as well as the intergenic region (IR) in the control of Tomato yellow leaf curl virus (TYLCV) in N. benthamiana. Interestingly, none of the novel variants of viruses observed in N. benthamiana was carrying sgRNAs targeting IR sequences. Further, targeted editing in the non-coding IR region of three geminiviruses, namely Cotton leaf curl Kokhran virus (CLCuKov), Melilotus mosaic virus (MeMV) and Tomato yellow leaf curl virus (TYLCV) had imparted resistance to multiple begomoviruses and simultaneously conferred broad-spectrum resistance against geminivirus [82].

Majority plant vclass="Gene">iruses have an RNA genome, which is usually single-stranded (ss) or double-stranded (ds) in single or multiclass="Chemical">ple fragments. Since the sgRNA-class="Chemical">pan class="Gene">Cas9 system only recognizes DNA sequences, it becomes difficult to achieve resistance against RNA viruses. However, there are some Cas9 variants, which have the potential to target and cleave RNA sequences, which need to be explored. Therefore, the CRISPR/CAS9 system has been utilized to derive resistance to RNA viruses, mainly through targeting host genes responsible for susceptibility. Chandrasekaran et al., 2016 [83] demonstrated the utility of CRISPR/CAS9 system in developing disease resistance in cucumber plants against Zucchini yellow mosaic virus, Cucumber vein yellowing virus and Papaya ringspot virus by targeted editing of eukaryotic translation initiation factor 4E (eIF4E). The RNA viruses specifically attach to eIF4E through virus-encoded movement protein (VPg) and a mutation in the host eIF4E limits the viral establishment. Similarly, resistance against Turnip mosaic virus (TuMV) has been achieved in A. thaliana by targeting eIF(iso)4 locus, which plays a vital role in viral survival [84]. Zhang et al. (2018) [85] expressed CRISPR-Cas9 system from Francisella novicida (FnCas9) in N. benthamiana and Arabidopsis plants to impart disease resistance against Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV). Macovei et al. (2018) [86] developed tungro disease resistance [caused by Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV)] in rice susceptible cultivar IR64 which was achieved by targeting translation initiation factor 4 gamma gene (eIF4G). Similarly, Aman et al. (2018) [87] exploited CRISPR/LshCas13a strategy to confer resistance against TuMV by targeting different viral genomic regions, namely helper component proteinase silencing suppressor (HC-Pro) and coat protein (CP) region. Further, a reduction in replication and spread of the virus was found to be efficient upon editing the HC-Pro region. Editing of host susceptibility factors to control RNA viruses requires comprehensive evaluation for understanding the fitness of edited plants in the field.

CRISclass="Chemical">PR based vclass="Chemical">pan class="Gene">irus control is a novel strategy, which can knock out a host factor required by the virus for the development of resistance. Moreover, virus resistance would be durable than dominant R genes due to lower selective pressures on the virus to evolve counter defense strategies [88]. Besides, eukaryotic viruses have developed mechanisms to circumvent RNAi through the expression of RNAi suppressors [89]. Further, targeting essential cis-acting regions of the viral genome might help to prevent the escape of modified viruses [90]. Despite its appeal, CRISPR/CAS9 technology develops some off-target effects, and most of the CRISPR/Cas systems were based on the challenge results of agro-inoculations. Even though this method resembled natural inoculations, the differences in virion load between these two inoculation methods have not been systematically evaluated. Thus, it is better to perform inoculation on test plants with their respective vectors [91]. Finally, the virus has the ability to evolve constantly, and successful technology has to match the speed of the virus adaptability for providing better and quicker solutions.

CRISPR/CAS9 and plant beneficial microbes

class="Chemical">Plant beneficial microbes are often found associated with the class="Chemical">plant rhizosclass="Chemical">phere. class="Chemical">pan class="Chemical">Few beneficial microbes are endophytes, which aid the host by improving efficient nutrient use of macronutrients (N, P), and micronutrients (Zn, Mn, Cu and Fe) [92]. Additionally, microbe-induced root architectural modifications also improve nutrient use efficiency. Microbes also participate in the acclimatization process of several abiotic, biotic stresses, osmoregulation, and carbon sequestration [93-97]. Accordingly, beneficial microbes are valuable candidates, which enhance crop productivity and participate in bi-directional interaction wherein plants provide carbon source and microbes assist the plant acclimatization to physiological stresses. CRISPR-Cas9 has been applied to understand soil microbiome-related processes, including nitrification and lignocellulose decomposition. Rice gene NRT1.1 B functions as nitrate transporter, which regulates the root microbiome in indica varieties [98]. Targeted allelic replacement of NRT1.1 in rice using CRISPR-Cas9 greatly enhanced nitrogen use efficiency in japonica rice, and the edited plants further served as biomarkers for understanding root microbiome. Thus, CRISPR technology is becoming a highly valuable tool for metagenomics assisted root microbiome studies. Additionally, CRISPR-Cas9 has been adopted for the alteration of plant cell wall components by specifically targeting OSH15 and OsAt10 genes for obtaining enhanced saccharification [99]. CRISPR elements are naturally present in microorganisms to provide anti-viral mechanism [100], and it was reported that polar soil has a higher abundance of CRISPR genes than tropical soil, which was correlated with greater disease pressure in the tropics [101]. Accordingly, horizontal gene transfers are greatly reduced in CRISPR-Cas9 edited Bacillus subtilis employing integrative plasmid wherein the risk of horizontal transfer could be lowered [102]. Hence, the presence of CRISPR repeats in plant beneficial bacteria may provide an evolutionary advantage for better adaptation.

CRISPR/CAS9 for mechanistic insights in symbiotic nitrogen fixation

Symbiotic class="Chemical">nitrogen fixation is one of the major beneficial legume-Rhizobium interactions in this world. The CRISclass="Chemical">pan class="Chemical">PR-Cas9 approach has been used to elucidate various genes involved in nitrogen fixation. Specifically, nodulation specific promoter region of leghemoglobin genes (LjLb1, LjLb2, LjLb3) was used for efficient expression of guideRNA in the nodules of Lotus japonica [103]. Further, the loss-of-function of cowpea (Vigna unguiculata) symbiotic receptor-like kinase (VuSYMRK) gene developed through CRISPR-Cas9 blocked the nodule formation [104]. In soybean, the dominant gene responsible for restriction of nodule formation by Sinorhizobium fredii was identified through the Rfg1 gene [105], and Rj4 gene [106] editing. Moreover, editing of four genes (PanHK4, PanEIN2, PanNSP1, and PanNSP2) related to hormonal regulation of nodulation in tropical tree, Parasponia andersonii, helped to identify conservation of nodulation process in comparison to that of legumes [107]. Recently, small fragments of tRNA (tRFs) synthesized in Rhizobia regulated 52 soybean genes were determined to be involved in the nodulation process. Further, CRISPR-Cas9 based editing of a few selected genes promoted nodulation in soybean [108]. In Medicago, CRISPR/CAS9 based editing of gibberlin oxidase (MtGA2Ox10) suppressed the infection thread formation in the initial nodulation process [109]. In addition, MtNFS2 gene responsible for strain specificity was identified and validated through the CRISPR/CAS9 approach in Medicago [103]. Thus, genome editing by CRISPR/CAS9 facilitates not only mechanistic insights in nodule formation but also assists in the identification of target genes, especially for enhancement of nodulation in legumes.

CRISPR/CAS9 approach in microbes

The CRISclass="Chemical">PR/class="Chemical">pan class="Gene">CAS9 system has already been reported in phytopathogens viz., Fusarium oxysporum, F. proliferatum, Alternaria alternata, Phytophthora spp. Sclerotinia sclerotiorum and Colletotrichum sansevieriae [110-114]. Specifically, F. proliferatum causes several diseases in plants and contributes to the production of diverse mycotoxins amongst which fumonisins are the most toxic. In Fusarium, FUM1 encodes a polyketide synthase gene responsible for the synthesis of fumonisins. CRISPR-Cas9 system was used to inactivate the FUM1 gene and the edited mutants did not produce fumonisins [115]. Further, the CRISPR-Cas9 tool has also been used to understand the infection process of fungal pathogens through the development of CRISPR-Cas9 assisted endogenous gene tagging (EGT) techniques. The EGT has been used to examine the infection process of F. oxysporum, wherein CRISPR-Cas9 was utilized for tagging endogenous genes with fluorescent markers providing an efficient tool for subcellular localization studies of fungal proteins [116]. Additionally, biocontrol fungus consists of clustered genes responsible for the production of secondary metabolites. Fang and Chen (2018) [117] demonstrated that the silencing of ace1 gene in Trichoderma atroviride induces the expression of four polyketide biosynthetic genes, which enhance the biocontrol activity against Fusarium oxysporum and Rhizoctonia solani. Hence, it is possible to enhance biocontrol potential by activating gene clusters through the CRISPR–Cas9 approach. Further, this approach could discover novel aspects of secondary metabolites pathways and also assists in the development of improved strains for better eco-friendly disease management.

Insights gained from using CRISPR/CAS9 approach in plant-microbe interactions

The recently developed CRISclass="Chemical">PR/class="Chemical">pan class="Gene">CAS9 technology has been so far extensively used in validating the previously identified genes/pathways involved in plant-microbe interactions. Additionally, the CRISPR/CAS9 approach has become one of the novel effective approaches for the control of viral diseases in crops. The differential disease response of novel alleles developed through genome editing in the resistance genes provides an indispensable tool in the hands of molecular breeders for crop improvement. Further, most of the genes studied so far either function at upstream or downstream components of plant-microbe interactions. However, none of the genes/pathways to our knowledge have been comprehensively understood from the initial pathogen interaction up to disease reaction in plants. In this regard, CRISPR/CAS9 based multiplex editing of target genes involved in a pathway has the potential to decipher the complete mechanistic understanding of plant-microbe interactions.

Future thrust

class="Chemical">Currently, CRISclass="Chemical">pan class="Chemical">PR/CAS9 is widely utilized for the development of durable disease resistance in plants. In most cases, the susceptibility gene(s) are edited for imparting durable disease resistance. However, nine different mechanisms were identified for resistance mediated through R genes in plants [118]. Therefore, targeting the genes involved in different pathways of resistance through the CRISPR/CAS9 approach could greatly assist in the development of durable resistance for multiple pathogens in crops. Besides durable resistance, editing of a few genes further resulted in broad-spectrum resistance. The applicability of broad-spectrum resistance in multiple crops will prove to be an interesting area of research in the near future. Additionally, the utilization of CRISPR/ Cas9 techniques in disease management through the induction of gene activation will provide novel genetic regulators for the control of plant diseases [119]. Pathogens are recognized by plants extracellularly and/or intracellularly, which was recently highlighted by Van der Burgh and Joosten (2019) [120] as a ‘spatial-immunity model’. Therefore, CRISPR-Cas9 based targeting of host genes localized in different organelles (spatial) involved in regulating colonization will provide multilayered resistance against diseases. There is a recent report on CRISPR/CAS9 based enhancement of nodulation by small fragments of tRNA and the loss-of-function of few genes enhanced nodulation in legumes. Hence, nodulation processes could be better understood through high-throughput editing system using pooled gRNA libraries specific for nodulation related genes [121]. Moreover, the CRISPR-Cas9 based editing system has been well established only in a few crops. However, future farming techniques incorporating climate resilience [122] necessitates the cultivation of minor cereals, vegetables and pulses. In this scenario, diseases are the major limiting factor for the diversification of crops. Thus, utilization of the CRISPR-Cas9 approach for the development of durable disease resistance in minor crops ought to be given high priority, especially considering climate resilience, the profitability of cultivation, food, and nutritional security.

Conclusion

The applications of CRISclass="Chemical">PR/class="Chemical">pan class="Gene">CAS9 in plant biology have exponentially increased in the last five years. The knowledge of plant-microbe interactions and the development of durable resistance in crops is a major research theme for scientists all over the world. The present review summarizes the utilization of genome editing (CRISPR/CAS9 ) tool targeting ~81 genes in plants and phytopathogens for understanding bacterial, fungal and viral disease interactions. Further, genes involved in the nodulation process were also highlighted. However, the development of durable resistance in crops is a highly challenging task. The initial insights gained in the understanding of plant-microbe interaction through the CRISPR/CAS9 approach will greatly assist in developing sustainable disease management strategies in the future.