Camilo Ernesto López

Identifying resistance gene analogs associated with resistances to different pathogens in common Bean

ABSTRACT A polymerase chain reaction approach using degenerate primers that targeted the conserved domains of cloned plant disease resistance genes (R genes) was used to isolate a set of 15 resistance gene analogs (RGAs) from common bean (Phaseolus vulgaris). Eight different classes of RGAs were obtained from nucleotide binding site (NBS)-based primers and seven from not previously described Toll/Interleukin-1 receptor-like (TIR)-based primers. Putative amino acid sequences of RGAs were significantly similar to R genes and contained additional conserved motifs. The NBS-type RGAs were classified in two subgroups according to the expected final residue in the kinase-2 motif. Eleven RGAs were mapped at 19 loci on eight linkage groups of the common bean genetic map constructed at Centro Internacional de Agricultura Tropical. Genetic linkage was shown for eight RGAs with partial resistance to anthracnose, angular leaf spot (ALS) and Bean golden yellow mosaic virus (BGYMV). RGA1 and RGA2 were associated with resistance loci to anthracnose and BGYMV and were part of two clusters of R genes previously described. A new major cluster was detected by RGA7 and explained up to 63.9% of resistance to ALS and has a putative contribution to anthracnose resistance. These results show the usefulness of RGAs as candidate genes to detect and eventually isolate numerous R genes in common bean.

Gene expression profile in response to Xanthomonas axonopodis pv. manihotis infection in cassava using a cDNA microarray.

A cassava cDNA microarray based on a large cassava EST database was constructed and used to study the incompatible interaction between cassava and Xanthomonas axonopodis pv. manihotis (Xam) strain CIO151. For microarray construction, 5700 clones from the cassava unigene set were amplified by polymerase chain reaction (PCR) and printed on glass slides. Microarray hybridization was performed using cDNA from cassava plants (resistant variety MBra685) collected at 12, 24, 48xa0h and 7 and 15xa0days post-infection as treatment and cDNA from mock-inoculated plants as control. A total of 199 genes were found to be differentially expressed (126 up-regulated and 73 down-regulated). A greater proportion of differentially-expressed genes was observed at 7xa0days after inoculation. Expression profiling and cluster analyses indicate that, in response to inoculation with Xam, cassava induces dozens of genes, including principally those involved in oxidative burst, protein degradation and pathogenesis-related (PR) genes. In contrast, genes encoding proteins that are involved in photosynthesis and metabolism were down regulated. In addition, various other genes encoding proteins with unknown function or showing no similarity to other proteins were also induced. Quantitative real time PCR experiments confirmed the reliability of our microarray data. In addition we showed that some genes are induced more rapidly in the resistant than in the susceptible cultivar.

Using cDNA and genomic sequences as tools to develop SNP strategies in cassava (Manihot esculenta Crantz)

Single nucleotide polymorphisms (SNP) are the most abundant type of DNA polymorphism found in animal and plant genomes. They provide an important new source of molecular markers that are useful in genetic mapping, map-based positional cloning, quantitative trait locus mapping and the assessment of genetic distances between individuals. Very little is known on the frequency of SNPs in cassava. We have exploited the recently-developed collection of cassava expressed sequence tags (ESTs) to detect SNPs in the five cultivars of cassava used to generate the sequences. The frequency of intra-cultivar and inter-cultivar SNPs after analysis of 111 contigs was one polymorphism per 905 and one per 1,032xa0bp, respectively; totaling 1 each 509xa0bp. We have obtained further information on the frequency of SNPs in six cassava cultivars by analysis of 33 amplicons obtained from 3′ EST and BAC end sequences. Overall, about 11xa0kb of DNA sequence was obtained for each cultivar. A total of 186 SNPs (136 and 50 from ESTs and BAC ends, respectively) were identified. Among these, 146 were intra-cultivar polymorphisms, while 80 were inter-cultivar polymorphisms. Thus the total frequency of SNPs was one per 62xa0bp. This information will help to develop new strategies for EST mapping as well as their association with phenotypic characteristics.

A unigene catalogue of 5700 expressed genes in cassava

Two economically important characters, starch content and cassava bacterial blight resistance, were targeted to generate a large collection of cassava ESTs. Two libraries were constructed from cassava root tissues of varieties with high and low starch contents. Other libraries were constructed from plant tissues challenged by the pathogen Xanthomonas axonopodis pv.manihotis. We report here the single pass sequencing of 11 954 cDNA clones from the 5’ ends, including 111 from the 3’ ends. Cluster analysis permitted the identification of a unigene set of 5700 sequences. Sequence analyses permitted the assignment of a putative functional category for 37% of sequences whereas ~ 16% sequences did not show any significant similarity with other proteins present in the database and therefore can be considered as cassava specific genes. A group of genes belonging to a large multigene family was identified. We characterize a set of genes detected only in infected libraries putatively involved in the defense response to pathogen infection. By comparing two libraries obtained from cultivars contrasting in their starch content a group of genes associated to starch biosynthesis and differentially expressed was identified. This is the first large cassava EST resource developed today and publicly available thus making a significant contribution to genomic knowledge of cassava.

Mapping EST-derived SSRs and ESTs involved in resistance to bacterial blight in Manihot esculenta

Cassava (Manihot esculenta Crantz) is a major root crop widely grown in the tropics. Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam), is an important disease in Latin America and Africa resulting in significant losses. The preferred control method is the use of resistant genotypes. Mapping expressed sequence tags (ESTs) and determining their co-localization with quantitative trait loci (QTLs) may give additional evidence of the role of the corresponding genes in resistance or defense. Twenty-one EST-derived simple sequence repeats (SSRs) were mapped in 16 linkage groups. ESTs showing similarities with candidate resistance genes or defense genes were also mapped using strategies such as restriction fragment length polymorphisms, cleaved amplified polymorphic sequences, and allele-specific primers. In total, 10 defense-related genes and 2 bacterial artificial chromosomes (BACs) containing resistance gene candidates (RGCs) were mapped in 11 linkage groups. Two new QTLs associated with resistance to Xam strains CIO121 and CIO151 were detected in linkage groups A and U, respectively. The QTL in linkage group U explained 61.6% of the phenotypic variance and was associated with an RGC-containing BAC. No correlation was found between the new EST-derived SSRs or other mapped ESTs and the new or previously reported QTLs.

Recent progress in the characterization of molecular determinants in the Xanthomonas axonopodis pv. manihotis-cassava interaction

Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam), is a widespread disease that affects cassava (Manihot esculenta Crantz). Studies on the pathogen population structure, pathogen diagnosis, identification and expression of plant genes involved in resistance have been carried out. Different molecular techniques were developed to assess the genetic diversity among the Xampopulations. Characterization of Xam population dynamics over time had enable us to determine the different factors that are associated with resistance breakdown and those that influence the genetic structure or virulence phenotypes of the pathogen’s population. Methods for detecting the pathogen in vegetative planting materials and true seeds were developed and contributed to reduce the impact of the disease. To better understand the genetics of resistance a quantitative trait loci (QTLs) approach was developed. Using a PCR-based strategy with degenerate primers we isolated two resistance gene candidates in cassava. We also characterized a region of a chromosome rich in R-gene like sequence. In this review we also report the main results obtained by transcript profiling methodologies, cDNA-AFLP and ESTs developed by the authors to characterize the genes involved in disease resistance. All together these techniques allowed the identification of molecular markers either associated to CBB resistance or that may represent putative genes involved in disease resistance. This article reviews current knowledge on the molecular cassava–Xam interactions.