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Xanthomonas species infect many important crops and cause huge yield loss. These pathogens deliver transcription activator-like (TAL) effectors into the cytoplasm of plant cells. TAL effectors move to host nuclei, directly bind to the promoters of host susceptible genes, and activate their transcription. However, the molecular mechanisms by which TAL effectors induce host transcription remain unclear. We herein demonstrated that TAL effectors interacted with the SIMILAR TO RCD ONE (SRO) family proteins OsSRO1a and OsSRO1b in nuclei. A transactivation assay using rice protoplasts indicated that OsSRO1a and OsSRO1b enhanced the activation of the OsSWEET14 promoter by the TAL effector AvrXa7. The AvrXa7-mediated expression of OsSWEET14 was significantly reduced in ossro1a mutants. However, the overexpression of OsSRO1a increased disease resistance by up-regulating the expression of defense-related genes, such as WRKY62 and PBZ1. This was attributed to OsSRO1a and OsSRO1b also enhancing the transcriptional activity of WRKY45, a direct regulator of WRKY62 expression. Therefore, OsSRO1a and OsSRO1b appear to positively contribute to transcription mediated by bacterial TAL effectors and rice transcription factors. [ABSTRACT FROM AUTHOR]

Strains of Xanthomonas citri pv. malvacearum cause bacterial blight of cotton, a potentially serious threat to cotton production worldwide, including in sub-Saharan countries. Development of disease symptoms, such as water soaking, has been linked to the activity of a class of type 3 effectors, called transcription activator-like (TAL) effectors, which induce susceptibility genes in the host's cells. To gain further insight into the global diversity of the pathogen, to elucidate their repertoires of TAL effector genes, and to better understand the evolution of these genes in the cotton-pathogenic xanthomonads, we sequenced the genomes of three African strains of X. citri pv. malvacearum using nanopore technology. We show that the cotton-pathogenic pathovar of X. citri is a monophyletic lineage containing at least three distinct genetic subclades, which appear to be mirrored by their repertoires of TAL effectors. We observed an atypical level of TAL effector gene pseudogenization, which might be related to resistance genes that are deployed to control the disease. Our work thus contributes to a better understanding of the conservation and importance of TAL effectors in the interaction with the host plant, which can inform strategies for improving resistance against bacterial blight in cotton. [ABSTRACT FROM AUTHOR]

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. A btl19 -13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.

Following photosynthesis, sucrose is translocated to sink organs, where it provides the primary source of carbon and energy to sustain plant growth and development. Sugar transporters from the SWEET (sugar will eventually be exported transporter) family are rate-limiting factors that mediate sucrose transport across concentration gradients, sustain yields, and participate in reproductive development, plant senescence, stress responses, as well as support plant-pathogen interaction, the focus of this study. We identified 25 SWEET genes in the walnut genome and distinguished each by its individual gene structure and pattern of expression in different walnut tissues. Their chromosomal locations, cis-acting motifs within their 5' regulatory elements, and phylogenetic relationship patterns provided the first comprehensive analysis of the SWEET gene family of sugar transporters in walnut. This family is divided into four clades, the analysis of which suggests duplication and expansion of the SWEET gene family in Juglans regia. In addition, tissue-specific gene expression signatures suggest diverse possible functions for JrSWEET genes. Although these are commonly used by pathogens to harness sugar products from their plant hosts, little was known about their role during Xanthomonas arboricola pv. juglandis (Xaj) infection. We monitored the expression profiles of the JrSWEET genes in different tissues of "Chandler" walnuts when challenged with pathogen Xaj417 and concluded that SWEET-mediated sugar translocation from the host is not a trigger for walnut blight disease development. This may be directly related to the absence of type III secretion system-dependent transcription activator-like effectors (TALEs) in Xaj417, which suggests different strategies are employed by this pathogen to promote susceptibility to this major aboveground disease of walnuts.

The bacterial plant pathogen Xanthomonas oryzae pv. oryzae is responsible for the foliar rice bacterial blight disease. Genetically contrasted, continent-specific, sublineages of this species can cause important damages to rice production both in Asia and Africa. We report on the genome of the CIX2779 strain of this pathogen, previously named NAI1 and originating from Niger. Oxford Nanopore long reads assembly and Illumina short reads polishing produced a genome sequence composed of a 4,725,792-bp circular chromosome and a 39,798-bp-long circular plasmid designated pCIX2779_1. The chromosome structure and base-level sequence are highly related to reference strains of African X. oryzae pv. oryzae and encode identical transcription activator-like effectors for virulence. Importantly, our in silico analysis strongly indicates that pCIX2779_1 is a genuine conjugative plasmid, the first indigenous one sequenced from an African strain of the X. oryzae species. [Graphic: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Xanthomonas oryzae pv. oryzae (Xoo) causes severe bacterial leaf blight (BLB) disease to many Vietnamese major rice cultivars, including the TBR225. OsSWEET13 belongs to group III of the OsSWEET gene family encoding sugar transport proteins, which is considered one of the "susceptibility" genes (S genes) necessary for BLB disease. In this study, the rice cultivar TBR225 was determined to be susceptible to 19/20 Vietnamese Xoo isolates collected from the Northern provinces. Two of the three tested isolates (VXO_60 and VXO_96 isolates) were shown to up-regulate OsSWEET13 upon the Xoo infection of the TBR225 cultivar. The TBR225 OsSWEET13 promoter was isolated for sequencing analysis. The isolated DNA fragment was 615 bp in size, contained an effector binding element (EBE) PthXo2 that was recognized by the type III-secretory transcription activator-like (TAL) proteins of the Xoo. This promoter showed a similarity of more than 99% to the published OsSWEET13 promoter sequences (AP014967.1 and CP018167.1). Our findings are basic for the generation of highyielding rice varieties with resistance to BLB disease by genetic engineering in Vietnam. [ABSTRACT FROM AUTHOR]

AvrHah1 [avirulence (avr) gene homologous to avrBs3 and hax2, no. 1] is a transcription activator-like (TAL) effector (TALE) in Xanthomonas gardneri that induces water-soaked disease lesions on fruits and leaves during bacterial spot of tomato. We observe that water from outside the leaf is drawn into the apoplast in X. gardneri-infected, but not X. gardneriΔavrHah1 (XgΔavrHah1)-infected, plants, conferring a dark, water-soaked appearance. The pull of water can facilitate entry of additional bacterial cells into the apoplast. Comparing the transcriptomes of tomato infected with X. gardneri vs. XgΔavrHah1 revealed the differential up-regulation of two basic helix-loop-helix (bHLH) transcription factors with predicted effector binding elements (EBEs) for AvrHah1. We mined our RNA-sequencing data for differentially up-regulated genes that could be direct targets of the bHLH transcription factors and therefore indirect targets of AvrHah1. We show that two pectin modification genes, a pectate lyase and pectinesterase, are targets of both bHLH transcription factors. Designer TALEs (dTALEs) for the bHLH transcription factors and the pectate lyase, but not for the pectinesterase, complement water soaking when delivered by XgΔavrHah1 By perturbing transcriptional networks and/or modifying the plant cell wall, AvrHah1 may promote water uptake to enhance tissue damage and eventual bacterial egression from the apoplast to the leaf surface. Understanding how disease symptoms develop may be a useful tool for improving the tolerance of crops from damaging disease lesions.

Xanthomonas oryzae pv. oryzae (Xoo) strains that cause bacterial leaf blight (BLB) limit rice (Oryza sativa) production and require breeding more resistant varieties. Transcription activator-like effectors (TALEs) activate transcription to promote leaf colonization by binding to specific plant host DNA sequences termed effector binding elements (EBEs). Xoo major TALEs universally target susceptibility genes of the SWEET transporter family. TALE-unresponsive alleles of clade III OsSWEET susceptibility gene promoter created with genome editing confer broad resistance on Asian Xoo strains. African Xoo strains rely primarily on the major TALE TalC, which targets OsSWEET14. Although the virulence of a talC mutant strain is severely impaired, abrogating OsSWEET14 induction with genome editing does not confer equivalent resistance on African Xoo. To address this contradiction, we postulated the existence of a TalC target susceptibility gene redundant with OsSWEET14. Bioinformatics analysis identified a rice locus named ATAC composed of the INCREASED LEAF INCLINATION 2 (ILI2) gene and a putative lncRNA that are shown to be bidirectionally upregulated in a TalC-dependent fashion. Gain-of-function approaches with designer TALEs inducing ATAC sequences did not complement the virulence of a Xoo strain defective for SWEET gene activation. While editing the TalC EBE at the ATAC loci compromised TalC-mediated induction, multiplex edited lines with mutations at the OsSWEET14 and ATAC loci remained essentially susceptible to African Xoo strains. Overall, this work indicates that ATAC is a probable TalC off-target locus but nonetheless documents the first example of divergent transcription activation by a native TALE during infection. [ABSTRACT FROM AUTHOR]

Summary: SWEETs play important roles in intercellular sugar transport. Induction of SWEET sugar transporters by Transcription Activator‐Like effectors (TALe) of Xanthomonas ssp. is key for virulence in rice, cassava and cotton.We identified OsSWEET11b with roles in male fertility and potential bacterial blight (BB) susceptibility in rice. While single ossweet11a or 11b mutants were fertile, double mutants were sterile. As clade III SWEETs can transport gibberellin (GA), a key hormone for spikelet fertility, sterility and BB susceptibility might be explained by GA transport deficiencies. However, in contrast with the Arabidopsis homologues, OsSWEET11b did not mediate detectable GA transport. Fertility and susceptibility therefore are likely to depend on sucrose transport activity.Ectopic induction of OsSWEET11b by designer TALe enabled TALe‐free Xanthomonas oryzae pv. oryzae (Xoo) to cause disease, identifying OsSWEET11b as a potential BB susceptibility gene and demonstrating that the induction of host sucrose uniporter activity is key to virulence of Xoo. Notably, only three of six clade III SWEETs are targeted by known Xoo strains from Asia and Africa.The identification of OsSWEET11b is relevant for fertility and for protecting rice against emerging Xoo strains that target OsSWEET11b. [ABSTRACT FROM AUTHOR]

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus bacterial canker (CBC), one of the most devastating citrus diseases. Most commercial citrus varieties are susceptible to CBC. However, some citrus varieties and wild citrus germplasms are CBC resistant and are promising in genetic increases in citrus resistance against CBC. We aimed to evaluate citrus germplasms for resistance against CBC. First, we developed a rapid evaluation method based on enhanced yellow fluorescent protein (eYFP)- labeled Xcc. The results demonstrated that eYFP does not affect the growth and virulence of Xcc. Xcc-eYFP allows measurement of bacterial titers but is more efficient and rapid than the plate colony counting method. Next, we evaluated citrus germplasms collected in China. Based on symptoms and bacterial titers, we identified that two citrus germplasms (Tchang' papeda and 'Huapi' kumquat) are resistant, whereas eight citrus germplasms ('Chongyi' wild mandarin, 'Mangshan' wild mandarin, 'Ledong' kumquat, 'Dali' citron. 'Yiliang' citron, 'Longyan' kumquat, 'Bawang' kumquat, and 'Daoxian' wild mandarin) are tolerant. In summary, we have developed a rapid evaluation method to test the resistance of citrus plants against CBC. This method was successfully used to identify two highly canker-resistant citrus germplasms and eight citrus germplasms with canker tolerance. These results could be leveraged in traditional breeding contexts or be used to identify canker resistance genes to increase the disease resistance of commercial citrus varieties via biotechnological approaches. [ABSTRACT FROM AUTHOR]

Summary: Transcription activator‐like (TAL) effectors are major virulence factors secreted by the type III secretion systems of Xanthomonas oryzae pv. oryzicola (Xoc) and X. oryzae pv. oryzae (Xoo), causing bacterial leaf streak and bacterial blight, respectively, in rice. However, the knowledge of Xoc TAL effector function in promoting bacterial virulence remains limited.Here, we isolated the highly virulent Xoc strain HGA4 from the outbreak region of Huanggang (Hubei, China), which contains four TAL effectors not found in the Chinese model strain RS105. Among these, Tal2b was selected for introduction into RS105, which resulted in a longer lesion length than that in the control.Tal2b directly binds to the promoter region of the gene and activates the expression of OsF3H03g, which encodes 2‐oxoglutarate‐dependent dioxygenase in rice. OsF3H03g negatively regulates salicylic acid (SA)‐related defense by directly reducing SA, and it plays a positive role in susceptibility to both Xoc and Xoo in rice.OsF3H03g interacts with a uridine diphosphate‐glycosyltransferase protein (OsUGT74H4), which positively regulates bacterial leaf streak susceptibility and may inactivate SA via glycosylation modification. [ABSTRACT FROM AUTHOR]

Abstract Rice (Oryza sativa L.) is a staple food crop, feeding more than 50% of the world’s population. Diseases caused by bacterial, fungal, and viral pathogens constantly threaten the rice production and lead to enormous yield losses. Bacterial blight (BB) and bacterial leaf streak (BLS), caused respectively by gram-negative bacteria Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), are two important diseases affecting rice production worldwide. Due to the economic importance, extensive genetic and genomic studies have been conducted to elucidate the molecular mechanism of rice response to Xoo and Xoc in the last two decades. A series of resistance (R) genes and their cognate avirulence and virulence effector genes have been characterized. Here, we summarize the recent advances in studies on interactions between rice and the two pathogens through these R genes or their products and effectors. Breeding strategies to develop varieties with durable and broad-spectrum resistance to Xanthomonas oryzae based on the published studies are also discussed.

Xanthomonas oryzae pv. oryzae (Xoo) possesses transcription activator-like (TAL) effectors, which are important virulence factors for bacterial blight disease. Rice nucleotide-binding leucine rich repeats receptor Xa1 recognizes TAL effectors, then induces various immune responses. To overcome host defenses, Xoo has developed TAL effector variants, termed interfering TAL (iTAL) (or truncated TAL) effectors. Japanese Xoo strain T7133 overcomes Xa1-mediated immunity. In this study, we determined the whole genome sequence of Xoo T7133 and identified TAL and iTAL effectors, revealing commonalities and differences in the TAL effectors between Xoo T7133 and avirulent strain T7174. In addition, Xoo T7133 has a different type of iTAL effector from Xoo T7174, suggesting that the iTAL effector of Xoo T7133 may contribute to the suppression of Xa1-mediated immunity. [ABSTRACT FROM AUTHOR]

Summary: Effective and durable disease resistance for bacterial blight (BB) of rice is a continuous challenge due to the evolution and adaptation of the pathogen, Xanthomonas oryzae pv. oryzae (Xoo), on cultivated rice varieties. Fundamental to this pathogens' virulence is transcription activator‐like (TAL) effectors that activate transcription of host genes and contribute differently to pathogen virulence, fitness or both. Host plant resistance is predicted to be more durable if directed at strategic virulence factors that impact both pathogen virulence and fitness. We characterized Tal7b, a minor‐effect virulence factor that contributes incrementally to pathogen virulence in rice, is a fitness factor to the pathogen and is widely present in geographically diverse strains of Xoo. To identify sources of resistance to this conserved effector, we used a highly virulent strain carrying a plasmid borne copy of Tal7b to screen an indica multi‐parent advanced generation inter‐cross (MAGIC) population. Of 18 QTL revealed by genome‐wide association studies and interval mapping analysis, six were specific to Tal7b (qBB‐tal7b). Overall, 150 predicted Tal7b gene targets overlapped with qBB‐tal7b QTL. Of these, 21 showed polymorphisms in the predicted effector binding element (EBE) site and 23 lost the EBE sequence altogether. Inoculation and bioinformatics studies suggest that the Tal7b target in one of the Tal7b‐specific QTL, qBB‐tal7b‐8, is a disease susceptibility gene and that the resistance mechanism for this locus may be through loss of susceptibility. Our work demonstrates that minor‐effect virulence factors significantly contribute to disease and provide a potential new approach to identify effective disease resistance. [ABSTRACT FROM AUTHOR]

Rice (Oryza sativa L.) is a staple food crop, feeding more than 50% of the world's population. Diseases caused by bacterial, fungal, and viral pathogens constantly threaten the rice production and lead to enormous yield losses. Bacterial blight (BB) and bacterial leaf streak (BLS), caused respectively by gram-negative bacteria Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), are two important diseases affecting rice production worldwide. Due to the economic importance, extensive genetic and genomic studies have been conducted to elucidate the molecular mechanism of rice response to Xoo and Xoc in the last two decades. A series of resistance (R) genes and their cognate avirulence and virulence effector genes have been characterized. Here, we summarize the recent advances in studies on interactions between rice and the two pathogens through these R genes or their products and effectors. Breeding strategies to develop varieties with durable and broad-spectrum resistance to Xanthomonas oryzae based on the published studies are also discussed. [ABSTRACT FROM AUTHOR]

The introduction of CRISPR/Cas systems has resulted in a strong impulse for the field of gene-targeted epigenome/epigenetic reprogramming (EpiEditing), where EpiEditors consisting of a DNA binding part for targeting and an enzymatic part for rewriting of chromatin modifications are applied in cells to alter chromatin modifications at targeted genome loci in a directed manner. Pioneering studies preceding this era indicated causal relationships of chromatin marks instructing gene expression. The accumulating evidence of chromatin reprogramming of a given genomic locus resulting in gene expression changes opened the field for mainstream applications of this technology in basic and clinical research. The growing knowledge on chromatin biology and application of EpiEditing tools, however, also revealed a lack of predictability of the efficiency of EpiEditing in some cases. In this perspective, the dependence of critical parameters such as specificity, effectivity, and sustainability of EpiEditing on experimental settings and conditions including the expression levels and expression times of the EpiEditors, their chromatin binding affinity and specificity, and the crosstalk between EpiEditors and cellular epigenome modifiers are discussed. These considerations highlight the intimate connection between the outcome of epigenome reprogramming and the details of the technical approaches toward EpiEditing, which are the main topic of this volume of Methods in Molecular Biology. Once established in a fully functional "plug-and-play" mode, EpiEditing will allow to better understand gene expression control and to translate such knowledge into therapeutic tools. These expectations are beginning to be met as shown by various in vivo EpiEditing applications published in recent years, several companies aiming to exploit the therapeutic power of EpiEditing and the first clinical trial initiated., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight of rice, depends on its type III secretion system and associated effector proteins to grow and colonize the vascular tissues of rice plants. The type III effectors include a family of closely related transcription activator-like (TAL) effectors and the rest of diverse effectors, so-called non-TAL effectors. Our understanding of non-TAL effectors for pathogenesis in rice blight is still limited. Here we report a feasible method to rapidly detect the activation of mitogen-activated protein kinase pathway in rice mesophyll protoplasts by the X. oryzae pv. oryzae derived peptidoglycan and screen for virulent effectors that can suppress the pathogen-associated molecular pattern triggered immunity (PTI) response. Amongst 17 non-TAL effectors transiently expressed in rice cells, we found that three effectors (XopZ, XopN, and XopV) were able to suppress the peptidoglycan-triggered MAPK activation. The triple mutant of the X. oryzae pv. oryzae strain PXO99A lacking XopZ, XopN, and XopV showed additively reduced virulence. Adding back either of genes restored the virulence of the triple mutant. Our results demonstrate the collective and redundant ability of defense suppression by non-TAL effectors in causing bacterial blight of rice.

Plants are vulnerable to disease through pathogen manipulation of phytohormone levels, which otherwise regulate development, abiotic, and biotic responses. Here, we show that the wheat pathogen Xanthomonas translucens pv. undulosa elevates expression of the host gene encoding 9-cis-epoxycarotenoid dioxygenase (TaNCED-5BS), which catalyzes the rate-limiting step in the biosynthesis of the phytohormone abscisic acid and a component of a major abiotic stress-response pathway, to promote disease susceptibility. Gene induction is mediated by a type III transcription activator-like effector. The induction of TaNCED-5BS results in elevated abscisic acid levels, reduced host transpiration and water loss, enhanced spread of bacteria in infected leaves, and decreased expression of the central defense gene TaNPR1. The results represent an appropriation of host physiology by a bacterial virulence effector. [ABSTRACT FROM AUTHOR]

Xanthomonas perforans is the predominant pathogen responsible for bacterial leaf spot of tomato and X. euvesicatoria for that of pepper in the southeast United States. Previous studies have indicated significant changes in the X. perforans population collected from Florida tomato fields over the span of 2 decades, including a shift in race and diversification into three phylogenetic groups driven by genome-wide homologousrecombination events derived from X. euvesicatoria. In our sampling of Xanthomonas strains associated with bacterial spot disease in Alabama, we were readily able to isolate X. perforans from symptomatic pepper plants grown in several Alabama counties, indicating a recent shift in the host range of the pathogen. To investigate the diversity of these pepper-pathogenic strains and their relation to populations associated with tomatoes grown in the southeast United States, we sequenced the genomes of eight X. perforans strains isolated from tomatoes and peppers grown in Alabama and compared them with previously published genome data available from GenBank. Surprisingly, reconstruction of the X. perforans core genome revealed the presence of two novel genetic groups in Alabama that each harbored a different transcription activation-like effector (TALE). While one TALE, AvrHah1, was associated with an emergent lineage pathogenic to both tomato and pepper, the other was identified as a new class within the AvrBs3 family, here designated PthXp1, and was associated with enhanced symptom development on tomato. Examination of patterns of homologous recombination across the larger X. euvesicatoria species complex revealed a dynamic pattern of gene flow, with multiple donors of Xanthomonas spp. associated with diverse hosts of isolation. [ABSTRACT FROM AUTHOR]

Xanthomonas oryzae pv. oryzae , the causal pathogen of bacterial blight of rice, depends on its type III secretion system and associated effector proteins to grow and colonize the vascular tissues of rice plants. The type III effectors include a family of closely related transcription activator-like (TAL) effectors and the rest of diverse effectors, so-called non-TAL effectors. Our understanding of non-TAL effectors for pathogenesis in rice blight is still limited. Here we report a feasible method to rapidly detect the activation of mitogen-activated protein kinase pathway in rice mesophyll protoplasts by the X. oryzae pv. oryzae derived peptidoglycan and screen for virulent effectors that can suppress the pathogen-associated molecular pattern triggered immunity (PTI) response. Amongst 17 non-TAL effectors transiently expressed in rice cells, we found that three effectors (XopZ, XopN, and XopV) were able to suppress the peptidoglycan-triggered MAPK activation. The triple mutant of the X. oryzae pv. oryzae strain PXO99A lacking XopZ , XopN , and XopV showed additively reduced virulence. Adding back either of genes restored the virulence of the triple mutant. Our results demonstrate the collective and redundant ability of defense suppression by non-TAL effectors in causing bacterial blight of rice. [ABSTRACT FROM AUTHOR]

Rice bacterial leaf blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years, BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TalF (formerly Tal5) or TalC, two important TALEs previously identified in West African Xoo, were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that talF and talC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either talC- or talF-unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TalF and TalC variants and provided a detailed insight into the diversity of TALE repertoires. All sequenced strains shared nine evolutionary related tal effector genes. Notably, a new TalF variant that is unable to induce OsSWEET14 was identified. Furthermore, two distinct TalB variants were shown to have lost the ability to simultaneously induce two susceptibility genes as previously reported for the founding members of this group from strains MAI1 and BAI3. Yet, both new TalB variants retained the ability to induce one or the other of the two susceptibility genes. These results reveal molecular and functional differences in tal repertoires and will be important for the sustainable deployment of broad-spectrum and durable resistance to BLB in West Africa.

Rice bacterial leaf blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years, BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TalF (formerly Tal5) or TalC, two important TALEs previously identified in West African Xoo , were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that talF and talC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either talC - or talF -unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TalF and TalC variants and provided a detailed insight into the diversity of TALE repertoires. All sequenced strains shared nine evolutionary related tal effector genes. Notably, a new TalF variant that is unable to induce OsSWEET14 was identified. Furthermore, two distinct TalB variants were shown to have lost the ability to simultaneously induce two susceptibility genes as previously reported for the founding members of this group from strains MAI1 and BAI3. Yet, both new TalB variants retained the ability to induce one or the other of the two susceptibility genes. These results reveal molecular and functional differences in tal repertoires and will be important for the sustainable deployment of broad-spectrum and durable resistance to BLB in West Africa. [ABSTRACT FROM AUTHOR]

Transcription activator-like effector (TALE)-dependent dominant disease resistance (R) genes in plants, also referred to as executor R genes, are induced on infection by phytopathogenic bacteria of the genus Xanthomonas harbouring the corresponding TALE genes. Unlike the traditional R proteins, the executor R proteins do not determine the resistance specificity and may function broadly in different plant species. The executor R gene Bs4C-R in the resistant genotype PI 235047 of the pepper species Capsicum pubescens (CpBs4C-R) confers disease resistance to Xanthomonas campestris pv. vesicatoria (Xcv) harbouring the TALE genes avrBsP/avrBs4. In this study, the synthetic genes of CpBs4C-R and two other Bs4C-like genes, the susceptible allele in the genotype PI585270 of C. pubescens (CpBs4C-S) and the CaBs4C-R homologue gene in the cultivar 'CM334' of Capsicum annum (CaBs4C), were characterized in tobacco (Nicotiana benthamiana) and rice (Oryza sativa). The Bs4C genes induced cell death in N. benthamiana. The functional Bs4C-eCFP fusion proteins were localized to the endoplasmic reticulum (ER) membrane in the leaf epidermal cells of N. benthamiana. The Xa10 promoter-Bs4C fusion genes in transgenic rice conferred strainspecific disease resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight in rice, and were specifically induced by the Xa10-incompatible Xoo strain PXO99A(pHM1avrXa10). The results indicate that the Bs4C proteins from pepper species function broadly in rice and the Bs4C protein-mediated cell death from the ER is conserved between dicotyledonous and monocotyledonous plants, which can be utilized to engineer novel and enhanced disease resistance in heterologous plants. [ABSTRACT FROM AUTHOR]

Xanthomonas spp. reduce crop yields and quality worldwide. During infection of their plant hosts, many strains secrete transcription activator-like (TAL) effectors, which enter the host cell nucleus and activate specific corresponding host genes at effector binding elements (EBEs) in the promoter. TAL effectors may contribute to disease by activating the expression of susceptibility genes or trigger resistance associated with the hypersensitive reaction (HR) by activating an executor resistance ( R) gene. The rice bacterial leaf streak pathogen X. oryzae pv. oryzicola (Xoc) is known to suppress host resistance, and no host R gene has been identified against it, despite considerable effort. To further investigate Xoc suppression of host resistance, we conducted a screen of effectors from BLS256 and identified Tal2a as an HR elicitor in rice when delivered heterologously by a strain of the closely related rice bacterial blight pathogen X. oryzae pv. oryzae (Xoo) or by the soybean pathogen X. axonopodis pv. glycines. The HR required the Tal2a activation domain, suggesting an executor R gene. Tal2a activity was differentially distributed among geographically diverse Xoc isolates, being largely conserved among Asian isolates. We identified four genes induced by Tal2a in next-generation RNA sequencing experiments and confirmed them using quantitative real-time reverse transcription-polymerase chain reaction (qPCR). However, neither individual nor collective activation of these genes by designer TAL effectors resulted in HR. A tal2a knockout mutant of BLS256 showed virulence comparable with the wild-type, but plasmid-based overexpression of tal2a at different levels in the wild-type reduced virulence in a directly corresponding way. Overall, the results reveal that host resistance suppression by Xoc plays a critical role in pathogenesis. Further, the dose-dependent avirulence activity of Tal2a and the apparent lack of a single canonical target that accounts for HR point to a novel, activation domain-dependent mode of action, which might involve, for example, a non-coding gene or a specific pattern of activation across multiple targets. [ABSTRACT FROM AUTHOR]

Summary In the past decade, Xenopus tropicalis has emerged as a powerful new amphibian genetic model system, which offers all of the experimental advantages of its larger cousin, Xenopus laevis. Here we investigated the efficiency of transcription activator-like effector nucleases (TALENs) for generating targeted mutations in endogenous genes in X. tropicalis. For our analysis we targeted the tyrosinase (oculocutaneous albinism IA) (tyr) gene, which is required for the production of skin pigments, such as melanin. We injected mRNA encoding TALENs targeting the first exon of the tyr gene into two-cell-stage embryos. Surprisingly, we found that over 90% of the founder animals developed either partial or full albinism, suggesting that the TALENs induced bi-allelic mutations in the tyr gene at very high frequency in the F0 animals. Furthermore, mutations tyr gene were efficiently transmitted into the F1 progeny, as evidenced by the generation of albino offspring. These findings have far reaching implications in our quest to develop efficient reverse genetic approaches in this emerging amphibian model.

Fluorescence microscopy imaging of specific chromosomal sites is essential for genome architecture research. To enable visualization of endogenous loci in mammalian cells, programmable DNA-binding proteins such as TAL effectors and CRISPR/dCas9 are commonly utilized. In addition, site-specific insertion of a TetO repeat array, coupled with TetR-enhanced green fluorescent protein fusion protein expression, can be used for labeling nonrepetitive endogenous loci. Here, we performed a comparison of several live-cell chromosome tagging methods, including their effect on subnuclear positioning, expression of adjacent genes, and DNA replication timing. Our results showed that the CRISPR-based imaging method can delay DNA replication timing and sister chromatid resolution at certain region. However, subnuclear localization of the labeled locus and gene expression from adjacent loci were unaffected by either TetO/TetR or CRISPR-based methods, suggesting that CRISPR-based imaging could be used for applications that do not require DNA replication analysis.

The rice pathogens Xanthomonas oryzae pathovar (pv.) oryzae and pv. oryzicola produce numerous transcription activator-like ( TAL) effectors that increase bacterial virulence by activating expression of host susceptibility genes. Rice resistance mechanisms against TAL effectors include polymorphisms that prevent effector binding to susceptibility gene promoters, or that allow effector activation of resistance genes. This study identifies, in the heirloom variety Carolina Gold Select, a third mechanism of rice resistance involving TAL effectors. This resistance manifests through strong suppression of disease development in response to diverse TAL effectors from both X. oryzae pathovars. The resistance can be triggered by an effector with only 3.5 central repeats, is independent of the composition of the repeat variable di-residues that determine TAL effector binding specificity, and is independent of the transcriptional activation domain. We determined that the resistance is conferred by a single dominant locus, designated Xo1, that maps to a 1.09 Mbp fragment on chromosome 4. The Xo1 interval also confers complete resistance to the strains in the African clade of X. oryzae pv. oryzicola, representing the first dominant resistance locus against bacterial leaf streak in rice. The strong phenotypic similarity between the TAL effector-triggered resistance conferred by Xo1 and that conferred by the tomato resistance gene Bs4 suggests that monocots and dicots share an ancient or convergently evolved mechanism to recognize analogous TAL effector epitopes. [ABSTRACT FROM AUTHOR]

Transcriptional activator like (TAL) effectors are important virulence factors of several plant pathogenic species of Xanthomonas . Members of this secreted protein family may activate transcription of; susceptibility factors, favoring disease, or resistance genes, triggering immunity. Accordingly, most research in this area has focused on the responses of a few model varieties of plants to the transcriptional activities of TAL effectors. However, a few studies suggest that plants may have evolved a diversity of additional responses to TAL effectors that are not well understood. These could include resistance to the effects of the TAL-activated susceptibility factors such as carbohydrate transporters, genomic multiplication of off-target effector binding sites, direct receptor recognition of the conserved TAL effector structure, or mutation of the host machinery required for TAL effector function. A better understanding of the diversity of plant responses to TAL effectors will be important for harnessing the potential of these proteins for agricultural applications. [ABSTRACT FROM AUTHOR]

Mutations in disease susceptibility (S) genes, here referred to as recessive resistance genes, have promise for providing broad durable resistance in crop species. However, few recessive disease resistance genes have been characterized. Here, we show that the broadly effective resistance gene xa5,for resistance to bacterial blight of rice ( Oryza sativa), is dependent on the effector genes present in the pathogen. Specifically, the effectiveness of xa5 in preventing disease by strains of Xanthomonas oryzae pv. oryzae is dependent on major transcription activation-like ( TAL) effector genes, and correlates with reduced expression of the cognate S gene s. xa5 is ineffective in preventing disease by strains containing the TAL effector gene pthXo1, which directs robust expression of the S gene Os SWEET11, a member of sucrose transporter gene family. Incompatibility is associated with major TAL effectors that target the known alternative S genes Os SWEET14 and Os SWEET13. Incompatibility is defeated by transfer of pthXo1 to otherwise xa5-incompatible strains or by engineering a synthetic designer TAL effector to boost SWEET gene expression. In either case, compatible or incompatible, target gene expression and lesion formation are reduced in the presence of xa5. The results indicate that xa5 functions as a quantitative trait locus, dampening effector function, and, regardless of compatibility, target gene expression. Resistance is hypothesized to occur when S gene expression, and, by inference, sucrose leakage, falls below a threshold level. [ABSTRACT FROM AUTHOR]

The bacterial plant pathogen Xanthomonas oryzae pv. oryzae is responsible for the foliar rice bacterial blight disease. Genetically contrasted, continent-specific, sublineages of this species can cause important damages to rice production both in Asia and Africa. We report on the genome of the CIX2779 strain of this pathogen, previously named NAI1 and originating from Niger. Oxford Nanopore long reads assembly and Illumina short reads polishing produced a genome sequence composed of a 4,725,792-bp circular chromosome and a 39,798-bp-long circular plasmid designated pCIX2779_1. The chromosome structure and base-level sequence are highly related to reference strains of African X. oryzae pv. oryzae and encode identical transcription activator-like effectors for virulence. Importantly, our in silico analysis strongly indicates that pCIX2779_1 is a genuine conjugative plasmid, the first indigenous one sequenced from an African strain of the X. oryzae species. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Transcription activator-like (TAL) effectors are type III-delivered transcription factors that enhance the virulence of plant pathogenic Xanthomonas species through the activation of host susceptibility (S) genes. TAL effectors recognize their DNA target(s) via a partially degenerate code, whereby modular repeats in the TAL effector bind to nucleotide sequences in the host promoter. Although this knowledge has greatly facilitated our power to identify new S genes, it can also be easily used to screen plant genomes for variations in TAL effector target sequences and to predict for loss-of-function gene candidates in silico. In a proof-of-principle experiment, we screened a germplasm of 169 rice accessions for polymorphism in the promoter of the major bacterial blight susceptibility S gene OsSWEET14, which encodes a sugar transporter targeted by numerous strains of Xanthomonas oryzae pv. oryzae. We identified a single allele with a deletion of 18 bp overlapping with the binding sites targeted by several TAL effectors known to activate the gene. We show that this allele, which we call xa41(t), confers resistance against half of the tested Xoo strains, representative of various geographic origins and genetic lineages, highlighting the selective pressure on the pathogen to accommodate OsSWEET14 polymorphism, and reciprocally the apparent limited possibilities for the host to create variability at this particular S gene. Analysis of xa41(t) conservation across the Oryza genus enabled us to hypothesize scenarios as to its evolutionary history, prior to and during domestication. Our findings demonstrate that resistance through TAL effector-dependent loss of S-gene expression can be greatly fostered upon knowledge-based molecular screening of a large collection of host plants. [ABSTRACT FROM AUTHOR]

Many pathovars of plant pathogenic bacteria Xanthomonas species inject transcription activator-like (TAL) effectors into plant host cells to promote disease susceptibility or trigger disease resistance. The rice TAL effector-dependent disease resistance gene Xa10 confers narrow-spectrum race-specific resistance to Xanthomonas oryzae pv. oryzae ( Xoo), the causal agent of bacterial blight disease in rice. To generate broad-spectrum and durable resistance to Xoo, we developed a modified Xa10 gene, designated as Xa10 E5. Xa10 E5 has an EBE-amended promoter containing 5 tandemly arranged EBEs each responding specifically to a corresponding virulent or avirulent TAL effector and a stable transgenic rice line containing Xa10 E5 was generated in the cultivar Nipponbare. The Xa10 E5 gene was specifically induced by Xoo strains that harbour the corresponding TAL effectors and conferred TAL effector-dependent resistance to the pathogens at all developmental stages of rice. Further disease evaluation demonstrated that the Xa10 E5 gene in either Nipponbare or 9311 genetic backgrounds provided broad-spectrum disease resistance to 27 of the 28 Xoo strains collected from 11 countries. The development of Xa10 E5 and transgenic rice lines provides new genetic materials for molecular breeding of rice for broad-spectrum and durable disease resistance to bacterial blight. [ABSTRACT FROM AUTHOR]