Alba E. Jofre-Garfias

The capsicum transcriptome DB: a "hot" tool for genomic research

Chili pepper (Capsicum annuum) is an economically important crop with no available public genome sequence. We describe a genomic resource to facilitate Capsicum annuum research. A collection of Expressed Sequence Tags (ESTs) derived from five C. annuum organs (root, stem, leaf, flower and fruit) were sequenced using the Sanger method and multiple leaf transcriptomes were deeply sampled using with GS-pyrosequencing. A hybrid assembly of 1,324,516 raw reads yielded 32,314 high quality contigs as validated by coverage and identity analysis with existing pepper sequences. Overall, 75.5% of the contigs had significant sequence similarity to entries in nucleic acid and protein databases; 23% of the sequences have not been previously reported for C. annuum and expand sequence resources for this species. A MySQL database and a user-friendly Web interface were constructed with search-tools that permit queries of the ESTs including sequence, functional annotation, Gene Ontology classification, metabolic pathways, and assembly information. The Capsicum Transcriptome DB is free available from http://www.bioingenios.ira.cinvestav.mx:81/Joomla/

Agrobacterium-mediated transformation of Amaranthus hypochondriacus: light- and tissue-specific expression of a pea chlorophyll a/b-binding protein promoter

Abstract Mature embryos of Amaranthus hypochondriacus (amaranth) were used to develop an in vitro culture system for plant regeneration and genetic transformation. Plants were regenerated from embryo-derived callus cultivated on Murashige and Skoog medium supplemented with 10 µM 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-2-methoxybenzoic acid and 10% coconut liquid endosperm. Transgenic plants were obtained by inoculation of mature embryo explants with a disarmed Agrobacterium strain containing the plasmid pGV2260(pEsc4), which carried the genes encoding neomycin phosphotransferase type II and β-glucuronidase. The presence of transgenes in the genome of transformed amaranth plants and their progeny was demonstrated by Southern blot hybridization. Tissue specific and light-inducible expression directed by a pea chlorophyll a/b-binding protein promoter was observed in transgenic amaranth plants and their progeny.

Plant regeneration from shoot apex explants of foxtail millet

Green and etiolated shoot apices of foxtail millet (Setaria italica L.) cv. Nese 2A were cultured on Murashige and Skoog medium with four concentrations of 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid. In all treatments, embryogenic calli capable of plant regeneration were induced after ten weeks in culture. Calli induced on 2 mg l-1 of 2,4-d from green apices gave a higher rate of plant regeneration in comparison with etiolated apices on the other treatments. Plant regeneration was obtained from one year-old cultures. Regenerated plants were successfully established in soil, reached maturity and produced seeds.

Callus and suspension culture induction, maintenance, and characterization.

Callus and cell suspension can be used for long-term cell cultures maintenance. This chapter describes procedures for the induction of somatic embryos of garlic, keeping a regeneration capacity for more than 5 yr, as well as the maintenance of a tobacco suspension culture (NT-1 cells), for more than 10 yr. Methods for plant regeneration and growth kinetics of garlic cultures are described, as well as for cell viability of NT-1 cells stained with 2,3,5 triphenyltetrazolium chloride. The packed cell volume determination as a parameter of growth is detailed.

The global arginine regulator ArgR controls expression of argF in Pseudomonas syringae pv. phaseolicola but is not required for the synthesis of phaseolotoxin or for the regulated expression of argK.

In Pseudomonas syringae pv. phaseolicola the enzyme ornithine carbamoyltransferase (OCTase), encoded by argF, is negatively regulated by argR, similar to what has been reported for Pseudomonas aeruginosa. However, production of the phaseolotoxin-resistant OCTase encoded by argK, synthesis of phaseolotoxin, and infectivity for bean pods occur independently of the ArgR protein.

First Report of Fragaria chiloensis cryptic virus, Fragaria chiloensis latent virus, Strawberry mild yellow edge virus, Strawberry necrotic shock virus, and Strawberry pallidosis associated virus in Single and Mixed Infections in Strawberry in Central Mexico

For phytosanitary purposes, the prevalence and incidence of viruses found in strawberry production within a centralized breeding program was investigated in Abasolo and Irapuato Counties, Guanajuato State, Mexico. Single and mixed infections of Strawberry mottle virus (SMoV) and Strawberry crinkle virus (SCV) were originally reported in the area (3), and subsequently, Strawberry latent ringspot virus (SLRSV) was also found (4). Samples of strawberry plants showing viral symptoms: stunting, mild chlorosis and reddening, occasional wrinkled, curled, and deformed leaves that may exhibit mottling, and chlorotic spots, forming a putative virus complex were collected in April and December 2007 and July and December 2008. The detection and identification of viruses reported in the United States, the country of origin of most of the imported plantlets, was carried out with sets of primers for 11 viruses, through reverse transcription (RT)-PCR (developed by Robert Martin and Ioannis Tzanetakis in Corvallis, OR). The endogenous NADH 2 subunit was employed to test the quality of the RNA extracted. Amplification conditions were: 40 cycles of 1 min at each temperature, denaturation at 95°C, annealing at 50°C for Strawberry necrotic shock virus (SNSV); 52°C for Strawberry mild yellow edge virus (SMYEV); 55°C for Fragaria chiloensis latent virus (FClLV), Strawberry pallidosis associated virus (SPaV), Fragaria chiloensis cryptic virus (FClCV), and SMoV; and 58°C for SCV and NADH dehydrogenase, followed by a final extension at 72°C of 5 min after completion of the 40 cycles. The cloning and nucleotide sequencing of amplified fragments revealed the presence of seven viral species in 40 samples collected. These were FClLV, SCV, SMoV, SNSV, SPaV, and SMYEV, which were allocated GenBank accession numbers of JQ629412, JQ629413, JQ629414, JQ629415, JQ629416, and JQ629417, respectively. Strawberry UC-4 and UC-10 (1,2) were planted as indicators of viral infections on an experimental plot. All seven viruses were detected in single or mixed infections. SMoV was the most commonly found in combination with other viruses. Out of 40 samples, 35 were positive for the presence of viruses and six had single infections, of which five had SMoV and one had SPaV. The remaining 29 samples had mixed infections with two or more viruses in a total of 22 combinations. The combination of FCICV + SMoV was present in five samples, whereas the combination of SMoV + SMYEV was in two samples. All other samples had two and up to six different viruses per plant. SMoV was detected in 26 out of the 40 samples tested. SNSV and FClCV were detected in 14 samples. SMYEV was present in 13 samples. SCV was present in nine samples, whereas SPaV and FClLV were found in eight samples each. To the best of our knowledge, this is the first report of FClLV, FClCV, SNSV, SMYEV, and SPaV in Mexico. References: (1) N. W. Frazier. Plant Dis. Rep. 58:28, 1974. (2) N. W. Frazier. Plant Dis. Rep. 58:203, 1974. (3) D. Teliz-Ortiz and A. Trejo-Reyes. Rev. Mex. Fitopatol. 7:38, 1989. (4) L. Pérez-Moreno et al. Rev. Mex. Fitopatol. 22:187, 2004.

Temperature, the GacS/GacA system, and host metabolites regulate the type VI secretion system of Pseudomonas syringae pv. phaseolicola

The recently discovered type VI secretion system (T6SS) is implicated in the pathogenic and/or virulence processes of diverse bacteria. The expression pattern of the T6SS differs among different organisms and also depends on several environmental factors. We initiated a study of the conditions that influence T6SS gene expression in Pseudomonas syringae pv. phaseolicola NPS3121. Our results indicate that low temperatures and plant extracts impact the expression of T6SS genes and that the process is subject to regulation by the GacS/GacA two-component system.