The overexpression of RXam1, a cassava gene coding for an RLK, confers disease resistance to Xanthomonas axonopodis pv. manihotis.

MAIN CONCLUSION: The overexpression of RXam1 leads to a reduction in bacterial growth of XamCIO136, suggesting that RXam1 might be implicated in strain-specific resistance. Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) is a prevalent disease in all regions, where cassava is cultivated. CBB is a foliar and vascular disease usually controlled through host resistance. Previous studies have found QTLs explaining resistance to several Xam strains. Interestingly, one QTL called XM5 that explained 13% of resistance to XamCIO136 was associated with a similar fragment of the rice Xa21-resistance gene called PCR250. In this study, we aimed to further identify and characterize this fragment and its role in resistance to CBB. Screening and hybridization of a BAC library using the molecular marker PCR250 as a probe led to the identification of a receptor-like kinase similar to Xa21 and were called RXam1 (Resistance to Xam 1). Here, we report the functional characterization of susceptible cassava plants overexpressing RXam1. Our results indicated that the overexpression of RXam1 leads to a reduction in bacterial growth of XamCIO136. This suggests that RXAM1 might be implicated in strain-specific resistance to XamCIO136.

Genomic survey of pathogenicity determinants and VNTR markers in the cassava bacterial pathogen Xanthomonas axonopodis pv. Manihotis strain CIO151.

Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of bacterial blight of cassava, which is among the main components of human diet in Africa and South America. Current information about the molecular pathogenicity factors involved in the infection process of this organism is limited. Previous studies in other bacteria in this genus suggest that advanced draft genome sequences are valuable resources for molecular studies on their interaction with plants and could provide valuable tools for diagnostics and detection. Here we have generated the first manually annotated high-quality draft genome sequence of Xam strain CIO151. Its genomic structure is similar to that of other xanthomonads, especially Xanthomonas euvesicatoria and Xanthomonas citri pv. citri species. Several putative pathogenicity factors were identified, including type III effectors, cell wall-degrading enzymes and clusters encoding protein secretion systems. Specific characteristics in this genome include changes in the xanthomonadin cluster that could explain the lack of typical yellow color in all strains of this pathovar and the presence of 50 regions in the genome with atypical nucleotide composition. The genome sequence was used to predict and evaluate 22 variable number of tandem repeat (VNTR) loci that were subsequently demonstrated as polymorphic in representative Xam strains. Our results demonstrate that Xanthomonas axonopodis pv. manihotis strain CIO151 possesses ten clusters of pathogenicity factors conserved within the genus Xanthomonas. We report 126 genes that are potentially unique to Xam, as well as potential horizontal transfer events in the history of the genome. The relation of these regions with virulence and pathogenicity could explain several aspects of the biology of this pathogen, including its ability to colonize both vascular and non-vascular tissues of cassava plants. A set of 16 robust, polymorphic VNTR loci will be useful to develop a multi-locus VNTR analysis scheme for epidemiological surveillance of this disease.

TALE1 from Xanthomonas axonopodis pv. manihotis acts as a transcriptional activator in plant cells and is important for pathogenicity in cassava plants.

Many plant-pathogenic bacteria suppress pathogen-associated molecular pattern (PAMP)-triggered immunity by injecting effector proteins into the host cytoplasm during infection through the type III secretion system (TTSS). This type III secretome plays an important role in bacterial pathogenicity in susceptible hosts. Xanthomonas axonopodis pv. manihotis (Xam), the causal agent of cassava bacterial blight, injects several effector proteins into the host cell, including TALE1(Xam) . This protein is a member of the Transcriptional Activator-Like effector (TALE) protein family, formerly known as the AvrBs3/PthA family. TALE1(Xam) has 13.5 tandem repeats of 34 amino acids each, as well as two nuclear localization signals and an acidic activation domain at the C-terminus. In this work, we demonstrate the importance of TALE1(Xam) in the pathogenicity of Xam. We use versions of the gene that lack different domains in the protein in structure-function studies to show that the eukaryotic domains at the 3' end are critical for pathogenicity. In addition, we demonstrate that, similar to the characterized TALE proteins from other Xanthomonas species, TALE1(Xam) acts as a transcriptional activator in plant cells. This is the first report of the identification of a TALE in Xam, and contributes to our understanding of the pathogenicity mechanisms employed by this bacterium to colonize and cause disease in cassava.

Análisis de expresión génica durante la respuesta de defensa de la yuca a la bacteriosis vascular (Añublo Bacteriano) / Gene expression analysis during cassava defense response to bacterial blight disease

La bacteriosis vascular de la yuca es una destructiva enfermedad en Sur América y África, causada por la bacteria Xanthomonas axonopodis pv. manihotis (Xam). Produce pérdidas entre el 12 por cien y 100 por cien de los cultivos. Algunos estudios se han realizado a nivel bioquímico y citoquímico para conocer las respuestas de defensa de la yuca a Xam; sin embargo,las bases moleculares de los mecanismos de defensa no han sido aún caracterizadas. Con el propósito de identificar genes diferencialmente expresados durante la respuesta de la planta al patógeno, se ha construido una librería sustractiva, usando el método de Sustracción Diferencial en Cadena (DSC), con 1536 clones de dos variedades resistentes(MBRA 685 y SG 107-35). De esta librería fueron seleccionados al azar 110 clones para ser secuenciados y realizar búsquedas de similitud en bases de datos públicas. El análisis de secuencia mostró 14 clones con similitud a genes previamente reportados como involucrados en procesos de defensa en plantas, 70 clones con similitud a genes de plantas sin función conocida o que no presentaron similitud, representando nuevos genes potencialmente involucrados en las respuestas de defensa de la yuca. Finalmente fueron construidos microarreglos de ADNc, usando los clones seleccionados de las librerías sustractivas para confirmar su expresión diferencial durante el desarrollo dela infección.

Limitaciones de la bacteriosis vascular de yuca: Nuevos avances / Limitations of Cassava Bacterial Blight: New Advances

La yuca (Manihot esculenta) constituye la base de la alimentación de más de 600 millones de personas en el mundo. Una de las principales limitaciones de este cultivo es la bacteriosis vascular, ocasionada por la bacteria Xanthomonas axonopodis pv. manihotis (Xam). Este artículo revisa el conocimiento actual acerca de la interacción Xanthomonasyuca. Se presentan estudios recientes llevados a cabo sobre la diversidad y dinámica de las poblaciones de Xam empleando diferentes estrategias moleculares. Se describen los diferentes métodos desarrollados para la detección y diagnóstico de la bacteria en plantas y semillas de yuca y su contribución para reducir el impacto de la enfermedad. Se presentan los estudios encaminados a comprender los mecanismos moleculares y los genes responsables en la resistencia de la yuca a la bacteriosis vascular incluyendo los últimos avances obtenidos gracias a la aplicación de estrategias de genómica funcional. El conocimiento adquirido en los últimos años en este patosistema permitirá desarrollar mejores estrategias para el manejo de la enfermedad así como desarrollar a corto plazo variedades de yuca resistentes a la bacteriosis lo que contribuiría a resolver uno de los principales problemas de los productores pobres de yuca y le abriría un horizonte promisorio al cultivo de la yuca en el mundo.

Genetic structure and population dynamics of Xanthomonas axonopodis pv. manihotis in Colombia from 1995 to 1999.

Restriction fragment length polymorphisms (RFLPs) were used to study the population genetics and temporal dynamics of the cassava bacterial pathogen Xanthomonas axonopodis pv. manihotis. The population dynamics were addressed by comparing samples collected from 1995 to 1999 from six locations, spanning four different edaphoclimatic zones (ECZs). Forty-five different X. axonopodis pv. manihotis RFLP types or haplotypes were identified between 1995 and 1999. High genetic diversity of the X. axonopodis pv. manihotis strains was evident within most of the fields sampled. In all but one site, diversity decreased over time within fields. Haplotype frequencies significantly differed over the years in all but one location. Studies of the rate of change of X. axonopodis pv. manihotis populations during the cropping cycle in two sites showed significant changes in the haplotype frequencies but not composition. However, variations in pathotype composition were observed from one year to the next at a single site in ECZs 1 and 2 and new pathotypes were described after 1997 in these ECZs, thus revealing the dramatic change in the pathogen population structure of X. axonopodis pv. manihotis. Disease incidence was used to show the progress of cassava bacterial blight in Colombia during the 5-year period in different ecosystems. Low disease incidence values were correlated with low rainfall in 1997 in ECZ 1.

Identification of genes in cassava that are differentially expressed during infection with Xanthomonas axonopodis pv. manihotis.

SUMMARY The cDNA-amplified fragment length polymorphism approach was used to identify differentially expressed transcripts from cassava infected by Xanthomonas axonopodis pv. manihotis (Xam). Approximately 3600 transcript-derived fragments (TDFs) were screened of which 340 were isolated. The nucleotide sequences of 250 TDFs were analysed and assembled into contigs and singletons. The amino acid sequences of their predicted products were compared with entries in databases and 63 of these clones showed homology to known plant genes. Of these, 32 showed similarity to plant defence proteins. Fifty-one TDFs corresponded to proteins of unknown function and 106 did not match any sequence in the public databases. Quantitative reverse transcription PCR was carried out with a selected set of gene transcripts that demonstrated an increase of expression during the infection. These results point out candidate genes that are associated with cassava resistance to Xam and reinforce the idea of a complex process occurring during this plant-pathogen interaction.

AFLP fingerprinting: an efficient technique for detecting genetic variation of Xanthomonas axonopodis pv. manihotis.

Xanthomonas axonopodis pv. manihotis (Xam) is the causative agent of cassava bacterial blight (CBB), a worldwide disease that is particularly destructive in South America and Africa. CBB is controlled essentially through the use of resistant varieties. To develop an appropriate disease management strategy, the genetic diversity of the pathogen's populations must be assessed. Until now, the genetic diversity of Xam was characterized by RFLP analyses using ribotyping, and plasmid and genomic Xam probes. We used AFLP (amplified fragment length polymorphism), a novel PCR-based technique, to characterize the genetic diversity of Colombian Xam isolates. Six Xam strains were tested with 65 AFLP primer combinations to identify the best selective primers. Eight primer combinations were selected according to their reproducibility, number of polymorphic bands and polymorphism detected between Xam strains. Forty-seven Xam strains, originating from different Colombian ecozones, were analysed with the selected combinations. Results obtained with AFLP are consistent with those obtained with RFLP, using plasmid DNA as a probe. Some primer combinations differentiated Xam strains that were not distinguished by RFLP analyses, thus AFLP fingerprinting allowed a better definition of the genetic relationships between Xam strains.