Deep sampling of the Palomero maize transcriptome by a high throughput strategy of pyrosequencing

<p>Abstract</p> <p>Background</p> <p>In-depth sequencing analysis has not been able to determine the overall complexity of transcriptional activity of a plant organ or tissue sample. In some cases, deep parallel sequencing of Expressed Sequence Tags (ESTs), although not yet optimized for the sequencing of cDNAs, has represented an efficient procedure for validating gene prediction and estimating overall gene coverage. This approach could be very valuable for complex plant genomes. In addition, little emphasis has been given to efforts aiming at an estimation of the overall transcriptional universe found in a multicellular organism at a specific developmental stage.</p> <p>Results</p> <p>To explore, in depth, the transcriptional diversity in an ancient maize landrace, we developed a protocol to optimize the sequencing of cDNAs and performed 4 consecutive GS20–454 pyrosequencing runs of a cDNA library obtained from 2 week-old <it>Palomero Toluqueño </it>maize plants. The protocol reported here allowed obtaining over 90% of informative sequences. These GS20–454 runs generated over 1.5 Million reads, representing the largest amount of sequences reported from a single plant cDNA library. A collection of 367,391 quality-filtered reads (30.09 Mb) from a single run was sufficient to identify transcripts corresponding to 34% of public maize ESTs databases; total sequences generated after 4 filtered runs increased this coverage to 50%. Comparisons of all 1.5 Million reads to the Maize Assembled Genomic Islands (MAGIs) provided evidence for the transcriptional activity of 11% of MAGIs. We estimate that 5.67% (86,069 sequences) do not align with public ESTs or annotated genes, potentially representing new maize transcripts. Following the assembly of 74.4% of the reads in 65,493 contigs, real-time PCR of selected genes confirmed a predicted correlation between the abundance of GS20–454 sequences and corresponding levels of gene expression.</p> <p>Conclusion</p> <p>A protocol was developed that significantly increases the number, length and quality of cDNA reads using massive 454 parallel sequencing. We show that recurrent 454 pyrosequencing of a single cDNA sample is necessary to attain a thorough representation of the transcriptional universe present in maize, that can also be used to estimate transcript abundance of specific genes. This data suggests that the molecular and functional diversity contained in the vast native landraces remains to be explored, and that large-scale transcriptional sequencing of a presumed ancestor of the modern maize varieties represents a valuable approach to characterize the functional diversity of maize for future agricultural and evolutionary studies.</p

EVALUACIÓN in vitro DE PRODUCTOS QUÍMICOS NO CONVENCIONALES PARA EL CONTROL DE Penicillium citrinum.

El ajo (Allium sativumL.), es un cultivo que esta expuesto al ataque de patógenos importantes del género Penicillium provocando pérdidas económicas importantes. De manera tradicional, la alternativa para controlar fitopatógenos es mediante la aplicación de fungicidas sintéticos. Sin embargo, debido a la aparición de cepas resistentes y problemas ambientales, es necesario la búsqueda de tratamientos alternativos que sean efectivos  y amigables con el medio ambiente. En este sentido, el uso de quitosano, peróxido de hidrógeno (H2O2), sorbato de potasio (SP) y bicarbonato de sodio (BS) representa una alternativa amigable con el medio ambiente con propiedades antimicrobianas. En este estudio, P. citrinum fue aislado e identificado mediante herramientas moleculares. En el ensayo in vitro, el H2O2al 0.5%, el BS al 3% y el quitosano al 1% resultaron efectivos en la inhibición del desarrollo del patógeno (crecimiento micelial, esporulación y germinación). Se observó un efecto sinérgico de los tratamientos evaluados en combinación a bajas concentraciones. Por lo tanto, los tratamientos evaluados pueden ser una alternativa viable y amigable con el medio ambiente el control de P. citrinumen ajo

A Functional Histidine-Tagged Replication Initiator Protein: Implications for the Study of Single-Stranded DNA Virus Replication In Planta

Replication initiation of nanoviruses, plant viruses with a multipartite circular single-stranded DNA genome, is triggered by the master Rep (M-Rep) protein. To enable the study of interactions between M-Rep and viral or host factors involved in replication, we designed oligohistidine-tagged variants of the nanovirus Faba bean necrotic yellows virus (FBNYV) M-Rep protein that allow affinity purification of enzymatically active M-Rep from plant tissue. The tagged M-Rep protein was able to initiate replication of its cognate and other FBNYV DNAs in Nicotiana benthamiana leaf disks and plants. The replicon encoding the tagged M-Rep protein multiplied and moved systemically in FBNYV-infected Vicia faba plants and was transmitted by the aphid vector of the virus. Using the tagged M-Rep protein, we demonstrated the in planta interaction between wild-type M-Rep and its tagged counterpart. Such a tagged and fully functional replication initiator protein will have bearings on the isolation of protein complexes from plants

Recognition of an Avr3a homologue plays a major role in mediating nonhost resistance to <em>Phytophthora capsici</em> in <em>Nicotiana</em> species

Nonhost resistance is a commonly occurring phenomenon wherein all accessions or cultivars of a plant species are resistant to all strains of a pathogen species and is likely the manifestation of multiple molecular mechanisms. Phytophthora capsici is a soil-borne oomycete that causes Phytophthora blight disease in many solanaceous and cucurbitaceous plants worldwide. Interest in P. capsici has increased considerably with the sequencing of its genome and its increasing occurrence in multiple crops. However, molecular interactions between P. capsici and both its hosts and its nonhosts are poorly defined. We show here that tobacco (Nicotiana tabacum) acts like a nonhost for P. capsici and responds to P. capsici infection with a hypersensitive response (HR). Furthermore, we have found that a P. capsici Avr3a-like gene (PcAvr3a1) encoding a putative RXLR effector protein produces a HR upon transient expression in tobacco and several other Nicotiana species. This HR response correlated with resistance in 19 of 23 Nicotiana species and accessions tested, and knock-down of PcAvr3a1 expression by host-induced gene silencing allowed infection of resistant tobacco. Our results suggest that many Nicotiana species have the capacity to recognize PcAvr3a1 via the products of endogenous disease resistance (R) genes and that this R gene–mediated response is a major component of nonhost resistance to P. capsici. </jats:p

EffHunter: A Tool for Prediction of Effector Protein Candidates in Fungal Proteomic Databases

Pathogens are able to deliver small-secreted, cysteine-rich proteins into plant cells to enable infection. The computational prediction of effector proteins remains one of the most challenging areas in the study of plant fungi interactions. At present, there are several bioinformatic programs that can help in the identification of these proteins; however, in most cases, these programs are managed independently. Here, we present EffHunter, an easy and fast bioinformatics tool for the identification of effectors. This predictor was used to identify putative effectors in 88 proteomes using characteristics such as size, cysteine residue content, secretion signal and transmembrane domains

Genome Sequence of Bacillus halotolerans Strain MS50-18A with Antifungal Activity against Phytopathogens, Isolated from Saline Soil in San Luís Potosí, Mexico

Bacillus halotolerans strain MS50-18A, isolated from saline soil, possesses antifungal activity toward root rot causal phytopathogens and has friendly interactions with the chili pepper plant. The draft genome sequence is 4.06 Mb in length and contains 4,215 genes. Genes related to glycine/betaine uptake and bacilysin biosynthesis are present, supporting its saline stress tolerance and antifungal activity

Draft Genome Sequence of Bacillus subtilis 2C-9B, a Strain with Biocontrol Potential against Chili Pepper Root Pathogens and Tolerance to Pb and Zn

Bacillus subtilis 2C-9B, obtained from the rhizosphere of wild grass, exhibits inhibition against root rot causal pathogens in Capsicum annuum, Pb and Zn tolerance, and plant growth promotion in medium supplemented with Pb. The genome of B. subtilis 2C-9B was sequenced and the draft genome assembled, with a length of 4,215,855 bp and 4,723 coding genes

Draft genome sequence of Bacillus velezensis 2A-2B strain: a rhizospheric inhabitant of Sporobolus airoides (Torr.) Torr., with antifungal activity against root rot causing phytopathogens

Abstract A Bacillus velezensis strain from the rhizosphere of Sporobolus airoides (Torr.) Torr., a grass in central-north México, was isolated during a biocontrol of phytopathogens scrutiny study. The 2A-2B strain exhibited at least 60% of growth inhibition of virulent isolates of phytopathogens causing root rot. These phytopathogens include Phytophthora capsici, Fusarium solani, Fusarium oxysporum and Rhizoctonia solani. Furthermore, the 2A-2B strain is an indolacetic acid producer, and a plant inducer of PR1, which is an induced systemic resistance related gene in chili pepper plantlets. Whole genome sequencing was performed to generate a draft genome assembly of 3.953 MB with 46.36% of GC content, and a N50 of 294,737. The genome contains 3713 protein coding genes and 89 RNA genes. Moreover, comparative genome analysis revealed that the 2A-2B strain had the greatest identity (98.4%) with Bacillus velezensis

Transcriptomic Analysis of Avocado Hass (Persea americana Mill) in the Interaction System Fruit-Chitosan-Colletotrichum

Avocado (Persea americana) is one of the most important crops in Mexico as it is the main producer, consumer, and exporter of avocado fruit in the world. However, successful avocado commercialization is often reduced by large postharvest losses due to Colletotrichum sp., the causal agent of anthracnose. Chitosan is known to have a direct antifungal effect and acts also as an elicitor capable of stimulating a defense response in plants. However, there is little information regarding the genes that are either activated or repressed in fruits treated with chitosan. The aim of this study was to identify by RNA-seq the genes differentially regulated by the action of low molecular weight chitosan in the avocado-chitosan-Colletotrichum interaction system. The samples for RNA-seq were obtained from fruits treated with chitosan, fruits inoculated with Colletotrichum and fruits both treated with chitosan and inoculated with the fungus. Non-treated and non-inoculated fruits were also analyzed. Expression profiles showed that in short times, the fruit-chitosan system presented a greater number of differentially expressed genes, compared to the fruit-pathogen system. Gene Ontology analysis of differentially expressed genes showed a large number of metabolic processes regulated by chitosan, including those preventing the spread of Colletotrichum. It was also found that there is a high correlation between the expression of genes in silico and qPCR of several genes involved in different metabolic pathways