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Induction of ProBs3 14EBE :GUSi by a wide range of Xanthomonas citri strains. Grapefruit leaves were infiltrated with bacterial suspension [5 3 10 8 colony-forming units (cfu)/mL] of Agrobacterium containing ProBs3 14EBE :GUSi in a binary vector, and infiltrated, 5 h later, with bacterial suspensions (adjusted to approximately 5 3 10 8 cfu/mL) of Xanthomonas citri strains or water. The infiltrated leaves were assayed for b-glucuronidase (GUS) by measuring 4-methyl umbelliferone (4-MU) fluorescence 5 days after inoculation, expressed as nanomoles 4-MU per minute per milligram protein. Data represent the mean 6 standard error (SE) of three independent experiments.
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Xanthomonas citri subsp. citri (X. citri), causal agent of citrus canker, uses Transcription Activator-Like Effectors (TALEs) as major pathogenicity factors. TALEs, which are delivered into plant cells through the type III secretion system (T3SS), interact with Effector Binding Elements (EBEs) in host genomes to activate expression of downstream su...
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... to accommodate multiple EBEs, each autonomously binding corresponding TALEs and mediating the transcription of a downstream coding sequence (R€ omer et al., 2009a). In an attempt to generate a promoter that would mediate the recognition of a broad range of X. citri strains, the Bs3 promoter was engineered to accommodate 14 different EBEs (Fig. S2, see Supporting Information). These included the four EBEs corresponding to PthA1-4, which are present in the Brazilian Xanthomonas strain Xcc306 used in these assays. The EBEs were designed according to the TALE code ( Boch et al., 2009;Moscou and Bogdanove, 2009) and correspond to X. citri TALEs deposited in protein databases or ...
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... TALEs that are compatible with EBEs of our engineered promoter. To test our assumption, we assayed ProBs3 14EBE :GUSi against a set of 20 X. citri strains collected worldwide (Table 1). To do so, the ProBs3 14EBE : GUSi construct was delivered transiently into grapefruit leaves and subsequently inoculated with one of 20 different X. citri strains (Fig. 2). We found that all X. citri strains, except one (strain 290 from Saudi Arabia), activated ProBs3 14EBE :GUSi in planta, suggesting that the promoter-activating strains have at least one TALE that activates the engineered promoter. Notably, X. citri strain 290 fails to produce typical canker symptoms in grapefruit and in all likelihood ...
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... are present in a given pathogen population. Yet, the implementation of EBEs is limited to TALEs for which sequence information is available. Analysis of our EBE-enriched promoter showed that 19 of 20 X. citri strains transcriptionally activated this promoter. In this assay, X. citri strain 290 was the exception and did not activate the promoter (Fig. 2). This observation is in agreement with previous studies showing that X. citri strain 290 from Saudi Arabia fails to produce typical canker symptoms in grapefruit and, in all likelihood, is unable to grow in grapefruit (Al-Saadi, 2005;Verniere et al., 1998). The finding that X. citri strain 290 also did not produce any canker lesions ...
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... to accommodate multiple EBEs, each autonomously binding corresponding TALEs and mediating the transcription of a downstream coding sequence (R€ omer et al., 2009a). In an attempt to generate a promoter that would mediate the recognition of a broad range of X. citri strains, the Bs3 promoter was engineered to accommodate 14 different EBEs (Fig. S2, see Supporting Information). These included the four EBEs corresponding to PthA1-4, which are present in the Brazilian Xanthomonas strain Xcc306 used in these assays. The EBEs were designed according to the TALE code ( Boch et al., 2009;Moscou and Bogdanove, 2009) and correspond to X. citri TALEs deposited in protein databases or ...
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... TALEs that are compatible with EBEs of our engineered promoter. To test our assumption, we assayed ProBs3 14EBE :GUSi against a set of 20 X. citri strains collected worldwide (Table 1). To do so, the ProBs3 14EBE : GUSi construct was delivered transiently into grapefruit leaves and subsequently inoculated with one of 20 different X. citri strains (Fig. 2). We found that all X. citri strains, except one (strain 290 from Saudi Arabia), activated ProBs3 14EBE :GUSi in planta, suggesting that the promoter-activating strains have at least one TALE that activates the engineered promoter. Notably, X. citri strain 290 fails to produce typical canker symptoms in grapefruit and in all likelihood ...
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... are present in a given pathogen population. Yet, the implementation of EBEs is limited to TALEs for which sequence information is available. Analysis of our EBE-enriched promoter showed that 19 of 20 X. citri strains transcriptionally activated this promoter. In this assay, X. citri strain 290 was the exception and did not activate the promoter (Fig. 2). This observation is in agreement with previous studies showing that X. citri strain 290 from Saudi Arabia fails to produce typical canker symptoms in grapefruit and, in all likelihood, is unable to grow in grapefruit (Al-Saadi, 2005;Verniere et al., 1998). The finding that X. citri strain 290 also did not produce any canker lesions ...
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The Gram-negative bacterium Xanthomonas translucens infects a wide range of gramineous plants with a notable impact on small grain cereals. However, genomics-informed intra-species population structure and virulence repertories of the pathogen have rarely been investigated. In this study, the complete genome sequences of seven X. translucens strain...
Citations
... Additionally, a potent transgenic approach to managing several xanthomonads has been devised through the development of "designer" pathogen-inducible synthetic constructs. An elicitor such as an avr gene, or a cognate R gene, is expressed under the control of an engineered promoter containing multiple effector binding elements (EBEs) that are targeted by different transcription activator-like effectors (TALEs); infection by bacteria containing the corresponding TALEs activates ETI and confers resistance (80,166,170,172). Similarly, guard NLRs such as ZAR1, which can indirectly recognize the 12 activity of multiple effectors via association with different effector-targeted kinases or analogous decoys, may also be exploited for multiplexed resistance (82,207). ...
Bacterial diseases are a constant threat to crop production globally. Current management strategies rely on an array of tactics, including improved cultural practices; application of bactericides, plant activators, and biocontrol agents; and use of resistant varieties when available. However, effective management remains a challenge, as the longevity of deployed tactics is threatened by constantly changing bacterial populations. Increased scrutiny of the impact of pesticides on human and environmental health underscores the need for alternative solutions that are durable, sustainable, accessible to farmers, and environmentally friendly. In this review, we discuss the strengths and shortcomings of existing practices and dissect recent advances that may shape the future of bacterial disease management. We conclude that disease resistance through genome modification may be the most effective arsenal against bacterial diseases. Nonetheless, more research is necessary for developing novel bacterial disease management tactics to meet the food demand of a growing global population. Expected final online publication date for the Annual Review of Phytopathology, Volume 60 is August 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... The repeat variable di-residues (RVDs) in every repeat in the central TALE region can specifically recognize one base of target DNA [28,29]. Based on the RVD-EBE recognition codes, artificial EBEs can be integrated into E genes to engineer broad and durable R genes for multiple Xanthomonas diseases [30][31][32]. A single modified E gene could realize the improvement of resistance to BB and BLS. ...
Bacterial blight (BB) and bacterial leaf streak (BLS), caused by phytopathogenic bacteria Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively, are the most serious bacterial diseases of rice, while blast, caused by Magnaporthe oryzae (M. oryzae), is the most devastating fungal disease in rice. Generating broad-spectrum resistance to these diseases is one of the key approaches for the sustainable production of rice. Executor (E) genes are a unique type of plant resistance (R) genes, which can specifically trap transcription activator-like effectors (TALEs) of pathogens and trigger an intense defense reaction characterized by a hypersensitive response in the host. This strong resistance is a result of programed cell death induced by the E gene expression that is only activated upon the binding of a TALE to the effector-binding element (EBE) located in the E gene promoter during the pathogen infection. Our previous studies revealed that the E gene Xa23 has the broadest and highest resistance to BB. To investigate whether the Xa23-mediated resistance is efficient against Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of BLS, we generated a new version of Xa23, designated as Xa23p1.0, to specifically trap the conserved TALEs from multiple Xoc strains. The results showed that the Xa23p1.0 confers broad resistance against both BB and BLS in rice. Moreover, our further experiment on the Xa23p1.0 transgenic plants firstly demonstrated that the E-gene-mediated defensive reaction is also effective against M. oryzae, the causal agent of the most devastating fungal disease in rice. Our current work provides a new strategy to exploit the full potential of the E-gene-mediated disease resistance in rice.
... Similarly, a total of 14 EBEs for distinct TALEs were introduced into the promoter of the pepper Bs3 gene and then fused to the Xanthomonas effector-encoding gene avrGf1, which can trigger an HR in orange. Transient transformation in citrus leaves showed that the engineered cassette conferred resistance to multiple X. citri strains (Shantharaj et al., 2017). Theoretically, the more EBEs added, the better the effect that may be expected for a broad resistance spectrum and durability. ...
Xanthomonas spp. colonize lots of host plants and cause huge loss worldwide. Transcription activation-like effectors (TALEs) are secreted by Xanthomonas and translocated into host cell to manipulate the expression of target genes, especially for Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) which cause bacterial blight (BB) and bacterial leaf streak (BLS) respectively in rice. In this review, we summarize the progress on studies about the interaction between Xanthomonas and host, which covers not only rice but also others. TALEs are not only the key factors making plants susceptible, they are also essential components in plant resistance. Characterization of TALEs and TALE-like proteins has improved our understanding of TALE evolution and promoted the development of gene editing tools. In addition, the interactions between TALEs and host have also provided strategies and possibilities for genetic engineering in crop improvement.
... On the contrary, another common method that has been exploited is the integration of designed EBEs into the promoter region of an E gene (or its alleles) in order to expand the resistance spectrum, or to obtain additional resistance to other pathogens by targeting the desirable TALEs [60][61][62]. A single naturally evolved E gene basically recognizes one TALE, and it often exhibits race-specific resistance. ...
Executor (E) genes comprise a new type of plant resistance (R) genes, identified from host–Xanthomonas interactions. The Xanthomonas-secreted transcription activation-like effectors (TALEs) usually function as major virulence factors, which activate the expression of the so-called “susceptibility” (S) genes for disease development. This activation is achieved via the binding of the TALEs to the effector-binding element (EBE) in the S gene promoter. However, host plants have evolved EBEs in the promoters of some otherwise silent R genes, whose expression directly causes a host cell death that is characterized by a hypersensitive response (HR). Such R genes are called E genes because they trap the pathogen TALEs in order to activate expression, and the resulting HR prevents pathogen growth and disease development. Currently, deploying E gene resistance is becoming a major component in disease resistance breeding, especially for rice bacterial blight resistance. Currently, the biochemical mechanisms, or the working pathways of the E proteins, are still fuzzy. There is no significant nucleotide sequence homology among E genes, although E proteins share some structural motifs that are probably associated with the signal transduction in the effector-triggered immunity. Here, we summarize the current knowledge regarding TALE-type avirulence proteins, E gene activation, the E protein structural traits, and the classification of E genes, in order to sharpen our understanding of the plant E genes.
... Wang et al. first reported the TALENs induced simultaneous mutations of three homoallele TaMLO (A1,B1,D1) homoeologs in the same wheat plant confers heritable broad-spectrum resistance to powdery mildew (Wang et al. 2014a, b). When the EBS site was added to the R gene promoter or avirulence protein promoter, the binding of TALE mediated resistance (Hutin et al. 2016;Shantharaj et al. 2017). A modified R gene promoter having a concatemer of EBE sites for TALEs from multiple strains of the pathogen could impart broad-spectrum resistance (Hummel et al. 2012;Zeng et al. 2015). ...
Agricultural losses due to plant pathogens is one of the most serious challenges faced worldwide. Breeding and introgression of R genes into susceptible cultivars are specific but are overcome by pathogen evolution. Genome sequencing and
annotation have led to the identification and characterization of specific genes/gene families responsible for pathogenesis
both in the host and pathogens. The most effective and sustainable way to manage plant pathogens and crop improvement
is to use genetic engineering tools, that utilize the traditional transgene technology along with the latest genome editing.
Amongst the various tools that has been developed, RNA interference has been widely used for regulating gene expression
and a large number of small RNAs have been reported to have bidirectional movement between the host and the pathogen.
Host-induced gene silencing technology is a powerful tool for protecting the host from pathogens which can be induced by
expressing dsRNA-producing constructs in the host. Targeted genome editing using artificial nucleases precisely modify
the genomes and CRISPR-Cas9 is the most commonly used nuclease for precise genome editing. These technologies have
gained wide popularity due to numerous successful examples with gene knockouts, knockdowns, modifications, repression/
silencing as well as activation of target genes in crop plants. In this review, we summarize the most recent strategies that
have been utilized for exploiting the host and pathogen genes for disease resistance in plants.
... To overcome the problem of the predicted lack of durability in native executor R genes, which are only equipped with single EBEs, scientists engineered executor R genes that are preceded by several tandem-arranged EBEs matching to a variety of TALEs present in corresponding Xanthomonas pathovars, providing broad-spectrum resistance [8][9][10]. The concept of equipping a single executor coding sequence with numerous tandemly arranged EBEs seems reminiscent of the aforementioned CRISPR-based depletion of multiple EBEs to provide broad-spectrum resistance. ...
Phytopathogenic bacteria inject effector proteins into plant host cells to promote disease. Plant resistance (R) genes encoding nucleotide-binding leucine-rich repeat (NLR) proteins mediate the recognition of functionally and structurally diverse microbial effectors, including transcription-activator like effectors (TALEs) from the bacterial genus Xanthomonas. TALEs bind to plant promoters and transcriptionally activate either disease-promoting host susceptibility (S) genes or cell death-inducing executor-type R genes. It is perplexing that plants contain TALE-perceiving executor-type R genes in addition to NLRs that also mediate the recognition of TALE-containing xanthomonads. We present recent findings on the evolvability of TALEs, which suggest that the native function of executors is not in plant immunity, but possibly in the regulation of developmentally controlled programmed cell death (PCD) processes.
... Science-based crop resistance improvement and disease tackling require a compilation of knowledge to understand the cellular and molecular bases of plant-pathogen interactions. As Xanthomonas pathogenicity notably relies on TALEs, knowledge on diversity and function of these effectors, in conjunction with resistance (R) proteins, are a cornerstone for resistance breeding in the affected crops [51][52][53][54]. Among the mechanisms of resistance associated with TALEs [55], recessive resistance is mediated by loss-of-susceptibility (LoS) alleles, where alternative alleles of an S gene that does not possess the EBE on the promoter sequence prevent the recognition and upregulation of the S gene (reviewed by [53]). ...
Cassava (Manihot esculenta Crantz) is a staple crop that feeds about 800 million people and supports the economy of low-income farmers in tropics. Due to its outstanding performance, cassava grows under a wide range of temperatures, soil pH, salinity degrees, and water availability, which is highly desirable for climate change adaptation. Biotic stresses affecting cassava include pests and pathogens, being viral and bacterial pathogens the most economically relevant. The cassava mosaic disease and the cassava brown streak disease are the two main viral cassava diseases, which cause important yield losses. Among bacterial pathogens, Xanthomonas phaseoli pv. manihotis (Xpm), causal agent of the Cassava Bacterial Blight (CBB), is the most relevant one. CBB is a vascular disease characterized by water-soaked angular leafspots, leaf blight, wilting, stem rotting, and dieback. Xpm relies on its effectors to subvert plant immunity and establish successful infections. These effectors include Xanthomonas outer proteins and Transcription Activator-Like effectors (TALEs). TALEs are bacterial effectors that, once translocated to the host cell, migrate to the nucleus and search for specific DNA motifs (effector binding element - EBE) by means of their repetitive central region. Once located onto the target EBE, the C-terminal region interacts with host transcription factors and recruits the RNA polymerase to initiate transcription of downstream genes. TALEs play key roles in Xpm pathogenesis, notably through the transcriptional activation of susceptibility (S) genes like the sugar transporter MeSWEET10a. Although it is known that TAL20Xam668 and TAL14Xam668 and their homologs are key factors that enhance water-soaking and in-planta bacterial growth (known as major virulence TALEs), little is known about TALE diversity among Xpm populations, the distribution of the major virulence TALEs, and other mechanisms used by Xpm to promote disease. In this thesis, we addressed these matters in three chapters: i) diversity and function of TALEs, ii) search for alternative S genes, and iii) development of a CRISPRi-based tool to study TALE biology. The study of TALE diversity and function revealed 13 novel variants in Xpm, including a variant with 22 repeats that activates the S gene MeSWEET10a. Moreover, we identified two potential cases of functional convergence. The relevance of these results lies in the description of new variants of major virulence TALEs and potential TALE-targeted hubs. The study of alternative S genes led to the identification of another SWEET sugar transporter, MeSWEET10e, that is transcriptionally activated by Xpm, potentially as a secondary target of TALE15 variants. Phylogenies indicate that Xpm TALEs and their linked capabilities to activate MeSWEETs come from at least two different ancestor genes (convergent evolution). These findings will be useful in the conception of Xpm-resistant cassava varieties. Finally, as we faced difficulties to mutate TALEs in some Xpm strains, we set a silencing platform based on CRISPR interference (CRISPRi), which could be extrapolated to other gene families and other Xanthomonas species. Three single guide RNAs (sgRNAs) were designed to silence Xpm TALEs, and our data show that most TALEs in a strain can be silenced by one individual sgRNA. CRISPRi silencing resulted in decreased symptoms and lower in-planta bacterial titers. Furthermore, plasmid-borne TALEs protected against sgRNAs were expressed in the strains undergoing silencing and allowed complementation. Implementation of this platform in Xanthomonas cassavae, a non-vascular pathogen of cassava, demonstrated for the first time the role of TALEs in this orphan pathosystem and suggested the role of MeSWEET10a as a target of Xc. Our study provides a useful CRISPRi platform to assess the role of TALEs during host colonization, and overall, for the functional analysis of multigene families in this species.
... Science-based crop resistance improvement and disease tackling require a compilation of knowledge to understand the cellular and molecular bases of plant-pathogen interactions. As Xanthomonas pathogenicity notably relies on TALEs, knowledge on diversity and function of these effectors, in conjunction with resistance (R) proteins, are a cornerstone for resistance breeding in the affected crops [51][52][53][54]. Among the mechanisms of resistance associated with TALEs [55], recessive resistance is mediated by loss-of-susceptibility (LoS) alleles, where alternative alleles of an S gene that does not possess the EBE on the promoter sequence prevent the recognition and upregulation of the S gene (reviewed by [53]). ...
Transcription activator-like effectors (TALEs) play a significant role for pathogenesis in several xanthomonad pathosystems. Xanthomonas phaseoli pv. manihotis (Xpm), the causal agent of Cassava Bacterial Blight (CBB), uses TALEs to manipulate host metabolism. Information about Xpm TALEs and their target genes in cassava is scarce, but has been growing in the last few years. We aimed to characterize the TALE diversity in Colombian strains of Xpm and to screen for TALE-targeted gene candidates. We selected eighteen Xpm strains based on neutral genetic diversity at a country scale to depict the TALE diversity among isolates from cassava productive regions. RFLP analysis showed that Xpm strains carry TALomes with a bimodal size distribution, and affinity-based clustering of the sequenced TALEs condensed this variability mainly into five clusters. We report on the identification of 13 novel variants of TALEs in Xpm, as well as a functional variant with 22 repeats that activates the susceptibility gene MeSWEET10a, a previously reported target of TAL20
Xam668
. Transcriptomics and EBE prediction analyses resulted in the selection of several TALE-targeted candidate genes and two potential cases of functional convergence. This study provides new bases for assessing novel potential TALE targets in the Xpm-cassava interaction, which could be important factors that define the fate of the infection.
... In another study, multiple effector-binding elements (EBEs) recognized by distinct Xanthomonas TAL effectors were introduced into the promoter of the pepper Bs3 resistance gene, and this engineered promoter was fused to the Xanthomonas avrGf1 gene, which encodes an avirulence protein that elicits an HR in grapefruit and sweet oranges (Shantharaj et al. 2017). Using transient expression assays, Shantharaj and colleagues (2017) found that the expression of avrGf1, induced by many X. ...
Citrus is one of the most ancient fruit crops cultivated in the world. For many countries, citrus production is an important source of revenues. However, despite the richness of citrus production worldwide, fruit yields are constantly threatened by diseases that cause serious economic and social impacts to growers and consumers. One such example is citrus canker, a disease that affects all commercial citrus varieties and for which there is no cure. Currently, control measures for citrus canker are restricted to the application of copper-based compounds and the elimination of infected trees to prevent pathogen spreading in the field. However, new alternatives for the control of this disease are being developed, spanning through transgenic plants to innovative chemicals and biological control. New genome editing approaches and a repertoire of plant defense-related genes have been exploited to produce citrus lineages more tolerant to X. citri. In addition, a series of new molecules have been tested to not only inhibit X. citri growth but to also boost the plant immune system to suppress disease progression. In this review, we provide the state of art of all citrus canker control measures in use today, highlighting their utility and drawbacks and commenting on novel strategies to better control this important citrus disease.
... Alternatively, EBEs can be added to the promoters of the resistance genes, which leads to the activation of resistance in the presence of TALEs. Researchers introduced 14 EBEs that match distinct X. citri TALEs into the ProBs3 14EBE promoter and fused it to the avirulence gene avrGf1, which induces a hypersensitive response in grapefruit and sweet orange 170 . Using resistance genes from closely related species to target single genes could lead to rapid development of pathogen virulence. ...
Xanthomonas spp. encompass a wide range of plant pathogens that use numerous virulence factors for pathogenicity and fitness in plant hosts. In this Review, we examine recent insights into host–pathogen co-evolution, diversity in Xanthomonas populations and host specificity of Xanthomonas spp. that have substantially improved our fundamental understanding of pathogen biology. We emphasize the virulence factors in xanthomonads, such as type III secreted effectors including transcription activator-like effectors, type II secretion systems, diversity resulting in host specificity, evolution of emerging strains, activation of susceptibility genes and strategies of host evasion. We summarize the genomic diversity in several Xanthomonas spp. and implications for disease outbreaks, management strategies and breeding for disease resistance.
