scholarly journals An efficient method to clone TAL effector genes from Xanthomonas oryzae using Gibson assembly

2019 ◽  
Vol 20 (10) ◽  
pp. 1453-1462 ◽  
Author(s):  
Chenhao Li ◽  
Chonghui Ji ◽  
José C. Huguet‐Tapia ◽  
Frank F. White ◽  
Hansong Dong ◽  
...  
Keyword(s):  
Efficient Method ◽  
Xanthomonas Oryzae ◽  
Gibson Assembly ◽  
Tal Effector ◽  
Effector Genes

scholarly journals Functional analysis of African Xanthomonas oryzae pv. oryzae TALomes reveals a new susceptibility gene in bacterial leaf blight of rice

2018 ◽  
Author(s):  
Tuan Tu Tran ◽  
Alvaro L Pérez-Quintero ◽  
Issa Wonni ◽  
Sara C. D. Carpenter ◽  
Yanhua Yu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
In Silico ◽  
Bacterial Blight ◽  
Xanthomonas Oryzae ◽  
Bacterial Leaf Blight ◽  
Genetic Lineage ◽  
Tal Effectors ◽  
Tal Effector ◽  
Effector Genes

AbstractMost Xanthomonas species translocate Transcription Activator-Like (TAL) effectors into plant cells where they function like plant transcription factors via a programmable DNA-binding domain. Characterized strains of rice pathogenic X. oryzae pv. oryzae harbor 9-16 different tal effector genes, but the function of only a few of them has been decoded. Using sequencing of entire genomes, we first performed comparative analyses of the complete repertoires of TAL effectors, herein referred to as TALomes, in three Xoo strains forming an African genetic lineage different from Asian Xoo. A phylogenetic analysis of the three TALomes combined with in silico predictions of TAL effector targets showed that African Xoo TALomes are highly conserved, genetically distant from Asian ones, and closely related to TAL effectors from the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Nine clusters of TAL effectors could be identified among the three TALomes, including three showing higher levels of variation in their repeat variable diresidues (RVDs). Detailed analyses of these groups revealed recombination events as a possible source of variation among TAL effector genes. Next, to address contribution to virulence, nine TAL effector genes from the Malian Xoo strain MAI1 and four allelic variants from the Burkinabe Xoo strain BAI3, thus representing most of the TAL effector diversity in African Xoo strains, were expressed in the TAL effector-deficient X. oryzae strain X11-5A for gain-of-function assays. Inoculation of the susceptible rice variety Azucena lead to the discovery of three TAL effectors promoting virulence, including two TAL effectors previously reported to target the susceptibility (S) gene OsSWEET14 and a novel major virulence contributor, TalB. RNA profiling experiments in rice and in silico prediction of EBEs were carried out to identify candidate targets of TalB, revealing OsTFX1, a bZIP transcription factor previously identified as a bacterial blight S gene, and OsERF#123, which encodes a subgroup IXc AP2/ERF transcription factor. Use of designer TAL effectors demonstrated that induction of either gene resulted in greater susceptibility to strain X11-5A. The induction of OsERF#123 by BAI3Δ1, a talB knockout derivative of BAI3, carrying these designer TAL effectors increased virulence of BAI3Δ1 validating OsERF#123 as a new, bacterial blight S gene.Author SummaryThe ability of most Xanthomonas plant pathogenic bacteria to infect their hosts relies on the action of a specific family of proteins called TAL effectors, which are transcriptional activators injected into the plant by the bacteria. TAL effectors enter the plant cell nucleus and bind to the promoters of specific plant genes. Genes that when induced can benefit pathogen multiplication or disease development are called susceptibility (S) genes. Here, we perform a comparative analysis of the TAL effector repertoires of three strains of X. oryzae pv. oryzae, which causes bacterial leaf blight of rice, a major yield constraint in this staple crop. Using sequencing of entire genomes, we compared the large repertoires of TAL effectors in three African Xoo strains which form a genetic lineage distinct from Asian strains. We assessed the individual contribution to pathogen virulence of 13 TAL effector variants represented in the three strains, and identified one that makes a major contribution. By combining host transcriptome profiling and TAL effector binding sites prediction, we identified two targets of this TAL effector that function as S genes, one previously identified, and one, new S gene. We validated the new S gene by functional characterization using designer TAL effectors. Both S genes encode transcription factors and can therefore be considered as susceptibility hubs for pathogen manipulation of the host transcriptome. Our results provide new insights into the diversified strategies underlying the roles of TAL effectors in promoting plant disease.

scholarly journals Colonization of Rice Leaf Blades by an African Strain of Xanthomonas oryzae pv. oryzae Depends on a New TAL Effector That Induces the Rice Nodulin-3 Os11N3 Gene

2011 ◽  
Vol 24 (9) ◽  
pp. 1102-1113 ◽  
Author(s):  
Yanhua Yu ◽  
Jana Streubel ◽  
Sandrine Balzergue ◽  
Antony Champion ◽  
Jens Boch ◽  
...  
Keyword(s):  
Specific Effect ◽  
Xanthomonas Oryzae ◽  
Upstream Sequence ◽  
In Planta ◽  
Tal Effectors ◽  
Disease Symptoms ◽  
Tal Effector ◽  
Glucuronidase Reporter ◽  
Reporter Assays ◽  
Family Based

African strains of Xanthomonas oryzae pv. oryzae contain fewer TAL effectors than Asian strains, and their contribution to pathogenicity is unknown. Systematic mutagenesis of tal genes was used to decipher the contribution of each of the eight TAL effector paralogs to pathogenicity of African X. oryzae pv. oryzae BAI3. A strain mutated in talC was severely affected in the production of disease symptoms. Analysis of growth in planta upon leaf-clip inoculation showed that mutant bacteria multiplied only at the site of inoculation at the apex of the leaf, suggesting a requirement for talC during colonization of vascular tissues. Such tissue-specific effect of a tal mutant is a novel phenotype, which has not yet been characterized in other xanthomonads. Microarray experiments comparing the host response of rice leaves challenged with BAI3R vs. BAI3RΔtalC were performed to identify genes targeted by TalC. A total of 120 upregulated and 21 downregulated genes were identified, among them Os11N3, which is a member of the MtN3/saliva family. Based on semiquantitative reverse transcription-polymerase chain reaction and β-glucuronidase reporter assays, we show that Os11N3 is directly upregulated by TalC and identify a TalC DNA target box within the Os11N3 upstream sequence.

scholarly journals An efficient method to clone TAL effector genes from Xanthomonas oryzae using Gibson assembly

2019 ◽  
Author(s):  
Chenhao Li ◽  
Chonghui Ji ◽  
José C. Huguet-Tapia ◽  
Frank F. White ◽  
Hansong Dong ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Critical Role ◽  
Repetitive Sequences ◽  
Xanthomonas Oryzae ◽  
Transcription Activator ◽  
Gibson Assembly ◽  
Vector Systems ◽  
Single Strand Annealing ◽  
Strand Annealing

AbstractTALes (Transcription Activator-Like effectors) represent the largest family of type III effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time-consuming due to multiple genes (up to 29 copies) in a given genome and technically challenging due to the repetitive sequences (up to 33 nearly identical 102-nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single strand annealing and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately one month compared to three months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest.

scholarly journals Rapid and efficient genome-wide characterization of Xanthomonas TAL effector genes

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Yan-Hua Yu ◽  
Ye Lu ◽  
Yong-Qiang He ◽  
Sheng Huang ◽  
Ji-Liang Tang
Keyword(s):  
Tal Effector ◽  
Effector Genes ◽  
Genome Wide

scholarly journals Mutagenesis of 18 Type III Effectors Reveals Virulence Function of XopZPXO99 in Xanthomonas oryzae pv. oryzae

2010 ◽  
Vol 23 (7) ◽  
pp. 893-902 ◽  
Author(s):  
Congfeng Song ◽  
Bing Yang
Keyword(s):  
Bacterial Blight ◽  
Xanthomonas Oryzae ◽  
Host Cells ◽  
Lesion Length ◽  
Transcription Activator ◽  
Tal Effectors ◽  
Type Iii ◽  
Effector Genes ◽  
Acid Protein ◽  
Bacterial Blight Of Rice

Xanthomonas oryzae pv. oryzae depends on a type III secretion system (T3SS) to translocate effectors into host cells for its ability to cause bacterial blight of rice. All type III (T3) effectors with known function in X. oryzae pv. oryzae belong to a family of transcription activator-like (TAL) effectors. However, other, non–TAL-related effector genes are present in the genome, although their role in virulence and their mode of action have yet to be elucidated. Here, we report the generation of mutants for 18 non-TAL T3 effector genes and the identification of one that contributes to the virulence of strain PXO99A. XopZPXO99 encodes a predicted 1,414-amino-acid protein of unknown function. PXO99A contains two identical copies of the gene due to a duplication of 212 kb in the genome. Strains with knockout mutations of one copy of XopZPXO99 did not exhibit any visible virulence defect. However, strains with mutations in both copies of XopZPXO99 displayed reduced virulence in terms of lesion length and bacterial multiplication compared with PXO99A. The introduction of one genomic copy of XopZPXO99 restores the mutant to full virulence. Transient expression of XopZPXO99 in Nicotiana benthamiana leaves suppresses host basal defense, which is otherwise induced by a T3SS mutant of PXO99A, suggesting a role for XopZPXO99 in interfering with host innate immunity during X. oryzae pv. oryzae infection. XopZPXO99-related genes are found in all Xanthomonas spp. whose genomic sequences have been determined, suggesting a conserved role for this type of effector gene in pathogenesis of Xanthomonas spp. Our results indicate that XopZPXO99 encodes a novel T3 effector and contributes virulence to X. oryzae pv. oryzae strains for bacterial blight of rice.

scholarly journals An efficient method for visualization and growth of fluorescent Xanthomonas oryzae pv. oryzae in planta

2008 ◽  
Vol 8 (1) ◽  
pp. 164 ◽  
Author(s):  
Sang-Wook Han ◽  
Chang-Jin Park ◽  
Sang-Won Lee ◽  
Pamela C Ronald
Keyword(s):  
Efficient Method ◽  
Xanthomonas Oryzae ◽  
In Planta

scholarly journals Resistance Genes and their Interactions with Bacterial Blight/Leaf Streak Pathogens (Xanthomonas oryzae) in Rice (Oryza sativa L.)—an Updated Review

Rice ◽  
2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Nan Jiang ◽  
Jun Yan ◽  
Yi Liang ◽  
Yanlong Shi ◽  
Zhizhou He ◽  
...  
Keyword(s):  
Oryza Sativa ◽  
Bacterial Blight ◽  
Oryza Sativa L ◽  
Rice Production ◽  
Xanthomonas Oryzae ◽  
Bacterial Leaf Streak ◽  
Viral Pathogens ◽  
Effector Genes ◽  
Broad Spectrum Resistance ◽  
Genomic Studies

AbstractRice (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.

scholarly journals Efficient enrichment cloning of TAL effector genes fromXanthomonas

2018 ◽  
Author(s):  
Tuan Tu Tran ◽  
Hinda Doucoure ◽  
Mathilde Hutin ◽  
Boris Szurek ◽  
Sebastien Cunnac ◽  
...  
Keyword(s):  
Target Genes ◽  
Protein Domain ◽  
Host Cells ◽  
Transcription Activator ◽  
Base Pairs ◽  
Tal Effectors ◽  
Tal Effector ◽  
Double Stranded Dna ◽  
Effector Genes ◽  
Gene Copies

Many plant-pathogenic xanthomonads use a type III secretion system to translocate Transcription Activator-Like (TAL) effectors into eukaryotic host cells where they act as transcription factors. Target genes are induced upon binding of a TAL effector to double-stranded DNA in a sequence-specific manner. DNA binding is governed by a highly repetitive protein domain, which consists of an array of nearly identical repeats of ca. 102 base pairs. Many species and pathovars ofXanthomonas, including pathogens of rice, cereals, cassava, citrus and cotton, encode multiple TAL effectors in their genomes. Some of the TAL effectors have been shown to act as key pathogenicity factors, which induce the expression of susceptibility genes to the benefit of the pathogen. However, due to the repetitive character and the presence of multiple gene copies, high-throughput cloning of TAL effector genes remains a challenge. In order to isolate complete TAL effector gene repertoires, we developed an enrichment cloning strategy based on (i) genome-informed in silico optimization of restriction digestions, (ii) selective restriction digestion of genomic DNA, and (iii) size fractionation of DNA fragments. Our rapid, cheap and powerful method allows efficient cloning of TAL effector genes from xanthomonads, as demonstrated for two rice-pathogenic strains ofXanthomonas oryzaefrom Africa.

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