Eduardo Canales

Genetic diversity among wild sugarcane germplasm from Laos revealed with markers

A group of 35 wild Saccharum complex clones, collected in Laos(ECL), was studied for its nuclear genetic diversity by RFLP revealed by 10dispersed low copy probes. Representatives of the main germplasmdiversity, regularly used in introgression, were also included. A preliminarycharacterisation of the new clones was performed by Erianthus specificprimers. A set of the 11 ECL clones most outstanding for their diseaseresistance and vigor and basic germplasm members were also characterisedby 9 cytoplasmic probes. Nuclear diversity evidenced that ECL clonesrepresented an independent genetic pool consisting at least of 3 differentgroups. A group of bands was exclusively exhibited by ECL clones. Theoccurrence of genetic recombination among them was suggested by thepresence of the new bands in 2 or 3 clonal groups. Erianthus specificamplification was positive for 7 ECL clones from different nuclear groups.Dendrogram analysis based on cytoplasmic diversity divided genotypes into2 major cytoplasmic types comprising 5 groups. The ECL clones separatedin 2 distinct groups: one located in the same cytoplasmic type as Erianthus clones; the other one, in an intermediate position, closer to Saccharum species. Basic clones groups were consistent with theirtaxonomic classification. The diversity revealed by nuclear singlecopy/nuclear repetitive/cytoplasmic patterns of the ECL germplasmcontrasts with what is usually observed and points to an active populationevolution. Further studies needed to confirmed this hypothesis arediscussed.

'Candidatus Liberibacter asiaticus', Causal Agent of Citrus Huanglongbing, Is Reduced by Treatment with Brassinosteroids.

Huanglongbing (HLB) constitutes the most destructive disease of citrus worldwide, yet no established efficient management measures exist for it. Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, development and stress tolerance. As a possible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, ‘Candidatus Liberibacter asiaticus’. The bacterial titers were reduced after treatment with epibrassinolide under both greenhouse and field conditions but were stronger in the greenhouse. Known defense genes were induced in leaves by epibrassinolide. With the SuperSAGE technology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified. The results demonstrate that epibrassinolide may provide a useful tool for the management of HLB.

Expression of a Nicotiana tabacum pathogen-induced gene is involved in the susceptibility to black shank

Many host genes induced during compatible plant-pathogen interactions constitute targets of pathogen virulence factors that act to suppress host defenses. In order to identify Nicotiana tabacum L. genes for pathogen-induced proteins involved in susceptibility to the oomycete Phytophthora parasitica var. nicotianae, we used SuperSAGE technology combined with next-generation sequencing to identify transcripts that were differentially upregulated during a compatible interaction. We identified a pathogen-induced gene (NtPIP) that was rapidly induced only during the compatible interaction. Virus-induced gene silencing of NtPIP reduced the susceptibility of N. tabacum to P. parasitica var. nicotianae. Additionally, transient expression of NtPIP in the resistant species Nicotiana megalosiphon Van Heurck & Mull. Arg. compromised the resistance to P. parasitica var. nicotianae. This pathogen-induced protein is therefore a positive regulator of the susceptibility response against an oomycete pathogen in tobacco.

Early regulation of primary metabolism, antioxidant, methyl cycle and phenylpropanoid pathways during the Mycosphaerella fijiensis-Musa spp. interaction

Black leaf streak disease (BLSD) caused by Mycosphaerella fijiensis is considered the most destructive and costly foliar disease that affects bananas and plantains. In spite of some recent progress regarding the study of M. fijiensis-Musa spp. interaction, there is still limited information for this pathosystem. To gain insights into the molecular mechanisms behind the M. fijiensis-Musa spp. interaction, gene expression and biochemical analysis related with primary metabolism, methyl cycle, antioxidant and phenylpropanoid pathways were conducted. Early screening of the resistant cultivar (cv.) ‘Calcutta 4’ (Musa AA) and the susceptible cv. ‘Grande naine’ (Musa AAA) plants infected with M. fijiensis was effective at looking for defense-related genes. Quantitative PCR experiments showed up-regulation of Photosystem I reaction center subunit N chloroplastic-like (primary metabolism) and S-adenosyl-L-methionine synthetaste (methyl cycle) genes in the incompatible interaction, as well as down-regulation of the phenylpropanoid pathway genes in the susceptible cv. ‘Grande naine’ as main finding of this study. Improved knowledge concerning the M. fijiensis-Musa spp. interaction could help to establish innovative approaches for plant breeding programs against BLSD.

NmEXT Extensin Gene: a Positive Regulator of Resistance Response Against the Oomycete Phytophthora nicotianae

The oomycete pathogens produce important diseases in many plant species. To identify extensin genes expressed during the oomycete Phytophthora nicotianae-Nicotiana megalosiphon interaction, we used the SuperSAGE technology. Using this approach, we detected a N. megalosiphon extensin gene (NmEXT) triggered during the interaction. The extensin gene accumulation induced by the pathogen correlated with disease resistance in different Nicotiana species. Transient expression of NmEXT gene in susceptible Nicotiana tabacum enhanced the resistance to P. nicotianae. Our date indicated that NmEXT gene served a positive role in N. tabacum resistance against P. nicotianae.

High-throughput SuperSAGE for gene expression analysis of Nicotiana tabacum – Rhizoctonia solani interaction

ObjectiveThe ubiquitous soil pathogen Rhizoctonia solani causes serious diseases in different plant species. Despite the importance of this disease, little is known regarding the molecular basis of susceptibility. SuperSAGE technology and next-generation sequencing were used to generate transcript libraries during the compatible Nicotiana tabacum–R. solani interaction. Also, we used the post-transcriptional silencing to evaluate the function of a group of important genes.ResultsA total of 8960 and 8221 unique Tag sequences identified as differentially up- and down-regulated were obtained. Based on gene ontology classification, several annotated UniTags corresponded to defense response, metabolism and signal transduction. Analysis of the N. tabacum transcriptome during infection identified regulatory genes implicated in a number of hormone pathways. Silencing of an mRNA induced by salicylic acid reduced the susceptibility of N. tabacum to R. solani. We provide evidence that the salicylic acid pathway was involved in disease development. This is important for further development of disease management strategies caused by this pathogen.

Cellular target of helminthosporium sacchari Toxin in sugarcane tissue

Spindles from sugarcane (Saccharum spp. hybrid) tissue were challenged using labeledHelminthosporium (Bipolaris) sacchari toxin to determine the general effect on different organelles and structures. Toxin labeling was characterized by biochemical and physiological behaviour of the fungal pathogen (dry weight, pH, refractive index, invertase and β -galactosidase activity and the biological activity of the toxin), allowing to improve the metabolic uptake of a labeled structural precursor of the toxin. A more efficient toxin labeling than previously reported was obtained. The labeled toxin was used as a tracer for determination of interactions at mitochondrial level. Binding activity reached saturation in time, confirming respiratory and ultrastructural analysis of the toxin effect.