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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.

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.

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.

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 Xanthomonasarboricola 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.

Xanthomonas spp are the causative agents of bacterial spot disease on cultivated pepper, Capsicum annuum, and tomato, Solanum lycopersicum. Although pepper and tomato are closely related in the Solanaceae, four species of xanthomonads have differing host specificities and utilize unique virulence strategies between these two crops plants. A major factor differentiating these pathogens are the Type III Effector (T3E) proteins they deploy to overcome the plant’s immune system and increase the host’s susceptibility. The genetic diversity and composition of T3E repertoires in a large sampling of field strains have yet to be explored on a genomic scale, limiting our understanding of pathogen evolution in an agricultural setting. To this end, we sequenced the genomes of sixty-seven Xanthomonas euvesicatoria (Xe), Xanthomonas perforans (Xp), and Xanthomonas gardneri (Xg) strains isolated from diseased pepper and tomato fields in the southeastern and midwestern United States. T3E repertoires were computationally predicted for each strain and whole genomic phylogenies were employed to understand better the genetic relationship of strains in the collection. From this analysis we detected a division in the Xp population that supported a model whereby a host-range expansion of Xp field strains on pepper is due, in part, to a loss of the T3E AvrBsT. Xp-host compatibility was further studied with the observation that a double deletion of the T3Es AvrBsT and XopQ allowed a gain of host range for Nicotiana benthamiana. Additionally, a single deletion of XopQ expanded the host range of Xe to N. benthamiana, while Xg was a natural pathogen of N. benthamiana. Extensive sampling of field strains and an improved understanding of effector content will aid in efforts to design plant disease resistance strategies targeted against highly conserved effectors.Xg has emerged recently as the dominant tomato pathogen in parts of the United States and South America. It is responsible for severe c

Xanthomonas spp are the causative agents of bacterial spot disease on cultivated pepper, Capsicum annuum, and tomato, Solanum lycopersicum. Although pepper and tomato are closely related in the Solanaceae, four species of xanthomonads have differing host specificities and utilize unique virulence strategies between these two crops plants. A major factor differentiating these pathogens are the Type III Effector (T3E) proteins they deploy to overcome the plant’s immune system and increase the host’s susceptibility. The genetic diversity and composition of T3E repertoires in a large sampling of field strains have yet to be explored on a genomic scale, limiting our understanding of pathogen evolution in an agricultural setting. To this end, we sequenced the genomes of sixty-seven Xanthomonas euvesicatoria (Xe), Xanthomonas perforans (Xp), and Xanthomonas gardneri (Xg) strains isolated from diseased pepper and tomato fields in the southeastern and midwestern United States. T3E repertoires were computationally predicted for each strain and whole genomic phylogenies were employed to understand better the genetic relationship of strains in the collection. From this analysis we detected a division in the Xp population that supported a model whereby a host-range expansion of Xp field strains on pepper is due, in part, to a loss of the T3E AvrBsT. Xp-host compatibility was further studied with the observation that a double deletion of the T3Es AvrBsT and XopQ allowed a gain of host range for Nicotiana benthamiana. Additionally, a single deletion of XopQ expanded the host range of Xe to N. benthamiana, while Xg was a natural pathogen of N. benthamiana. Extensive sampling of field strains and an improved understanding of effector content will aid in efforts to design plant disease resistance strategies targeted against highly conserved effectors.Xg has emerged recently as the dominant tomato pathogen in parts of the United States and South America. It is responsible for severe c

This work provides a comprehensive study of the molecular events mediated by the transcription activator-like (TAL) effectors of Xanthomonas axonopodis pv. manihotis (Xam) during infection of its host plant cassava (Manihot esculenta). TAL effectors are secreted via the bacterial type III secretion system into plant cells where they localize to the nucleus, bind specific sequences of nucleotides in plant promoters, and activate the expression of downstream genes. The DNA binding activity of Xam TAL effectors is carried out by a central domain containing a variable number of 34 amino acid repeats, where the 12th and 13th amino acid residues, the repeat variable diresidues (RVDs), of each repeat dictate which nucleotide will be bound at that site. Genetic analysis of the TAL effectors of highly virulent Xam strain Xam668 showed a virulence role for TAL20Xam668, which contributes to in planta bacterial growth and watersoaking symptom development, and TAL14Xam668, which contributes to in planta bacterial growth. RNA-Sequencing (RNA-Seq) revealed a single host gene target of TAL20Xam668, MeSWEET10a, which is a member of the SWEET family of sugar transporters. Designer TAL effectors that activate MeSWEET10a complemented the Xam668∆TAL20 mutant phenotype, revealing the role of MeSWEET10a in cassava’s susceptibility to Xam. SWEETs are known X. oryzae pv. oryzae TAL effector-targeted susceptibility genes in rice and thus this result establishes the activation of SWEET sugar transporters as a common mechanism of disease promotion for diverse species of Xanthomonas. In contrast to TAL20Xam668 which has a single target gene, RNA-Seq showed that TAL14Xam668 activates over 50 genes during the infection process. A subset of the TAL14Xam668-targeted genes were tested for activation by TAL14CIO151 from Xam strain CIO151 and, although TAL14CIO151 and TAL14Xam668 differ by only a single RVD, they display differential activation of host gene targets. TAL14CIO151 complements the TAL14Xa

This work provides a comprehensive study of the molecular events mediated by the transcription activator-like (TAL) effectors of Xanthomonas axonopodis pv. manihotis (Xam) during infection of its host plant cassava (Manihot esculenta). TAL effectors are secreted via the bacterial type III secretion system into plant cells where they localize to the nucleus, bind specific sequences of nucleotides in plant promoters, and activate the expression of downstream genes. The DNA binding activity of Xam TAL effectors is carried out by a central domain containing a variable number of 34 amino acid repeats, where the 12th and 13th amino acid residues, the repeat variable diresidues (RVDs), of each repeat dictate which nucleotide will be bound at that site. Genetic analysis of the TAL effectors of highly virulent Xam strain Xam668 showed a virulence role for TAL20Xam668, which contributes to in planta bacterial growth and watersoaking symptom development, and TAL14Xam668, which contributes to in planta bacterial growth. RNA-Sequencing (RNA-Seq) revealed a single host gene target of TAL20Xam668, MeSWEET10a, which is a member of the SWEET family of sugar transporters. Designer TAL effectors that activate MeSWEET10a complemented the Xam668∆TAL20 mutant phenotype, revealing the role of MeSWEET10a in cassava’s susceptibility to Xam. SWEETs are known X. oryzae pv. oryzae TAL effector-targeted susceptibility genes in rice and thus this result establishes the activation of SWEET sugar transporters as a common mechanism of disease promotion for diverse species of Xanthomonas. In contrast to TAL20Xam668 which has a single target gene, RNA-Seq showed that TAL14Xam668 activates over 50 genes during the infection process. A subset of the TAL14Xam668-targeted genes were tested for activation by TAL14CIO151 from Xam strain CIO151 and, although TAL14CIO151 and TAL14Xam668 differ by only a single RVD, they display differential activation of host gene targets. TAL14CIO151 complements the TAL14Xa