Genome-Wide Analysis of the LRR-RLP Gene Family in a Wild Banana (Musa acuminata ssp. malaccensis) Uncovers Multiple Fusarium Wilt Resistance Gene Candidates.

Banana is the most popular fruit in the world, with a relevant role in food security for more than 400 million people. However, fungal diseases cause substantial losses every year. A better understanding of the banana immune system should facilitate the development of new disease-resistant cultivars. In this study, we performed a genome-wide analysis of the leucine-rich repeat receptor-like protein (LRR-RLP) disease resistance gene family in a wild banana. We identified 78 LRR-RLP genes in the banana genome. Remarkably, seven MaLRR-RLPs formed a gene cluster in the distal part of chromosome 10, where resistance to Fusarium wilt caused by Foc race 1 has been previously mapped. Hence, we proposed these seven MaLRR-RLPs as resistance gene candidates (RGCs) for Fusarium wilt. We also identified seven other banana RGCs based on their close phylogenetic relationships with known LRR-RLP proteins. Moreover, phylogenetic analysis of the banana, rice, and Arabidopsis LRR-RLP families revealed five major phylogenetic clades shared by these plant species. Finally, transcriptomic analysis of the MaLRR-RLP gene family in plants treated with Foc race 1 or Foc TR4 showed the expression of several members of this family, and some of them were upregulated in response to these Foc races. Our study provides novel insights into the structure, distribution, evolution, and expression of the LRR-RLP gene family in bananas as well as valuable RGCs that will facilitate the identification of disease resistance genes for the genetic improvement of this crop.

Universal Primers for Detection of Novel Plant Capsid-Less Viruses: Papaya Umbra-like Viruses as Example.

For diagnosis of positive-sense single-stranded RNA viruses, primers are usually raised against the sequence encoding capsid proteins, since structural proteins are more conserved. This chapter focuses on the design of primers for a group of novel viruses lacking a capsid, known as papaya Umbra-like viruses (unassigned genus) associated with Papaya Sticky Disease, which represent a threat to papaya production. Based on sequence alignments of a region encoding the RNA-dependent RNA Polymerase, universal primers to detect all the known viruses from four countries are proposed. The Forward universal primer can be used in combination with clade- and subclade-specific primers for rapid virus identification. We walk the reader through downloading sequences from nucleotide databases, doing sequence alignments and phylogenetic tree construction to identify conserved and variable regions as valid primer targets; we also show how to design and analyze the primers.

Data mining of metagenomes to find novel enzymes: a non-computationally intensive method.

Currently, there is a need of non-computationally-intensive bioinformatics tools to cope with the increase of large datasets produced by Next Generation Sequencing technologies. We present a simple and robust bioinformatics pipeline to search for novel enzymes in metagenomic sequences. The strategy is based on pattern searching using as reference conserved motifs coded as regular expressions. As a case study, we applied this scheme to search for novel proteases S8A in a publicly available metagenome. Briefly, (1) the metagenome was assembled and translated into amino acids; (2) patterns were matched using regular expressions; (3) retrieved sequences were annotated; and (4) diversity analyses were conducted. Following this pipeline, we were able to identify nine sequences containing an S8 catalytic triad, starting from a metagenome containing 9,921,136 Illumina reads. Identity of these nine sequences was confirmed by BLASTp against databases at NCBI and MEROPS. Identities ranged from 62 to 89% to their respective nearest ortholog, which belonged to phyla Proteobacteria, Actinobacteria, Planctomycetes, Bacterioidetes, and Cyanobacteria, consistent with the most abundant phyla reported for this metagenome. All these results support the idea that they all are novel S8 sequences and strongly suggest that our methodology is robust and suitable to detect novel enzymes.

A novel salt-inducible CrGPDH3 promoter of the microalga Chlamydomonas reinhardtii for transgene overexpression.

The expression of transgenes in the nucleus is an attractive alternative for the expression of recombinant proteins in the green microalga Chlamydomonas reinhardtii. For this purpose, a strong inducible promoter that allows protein accumulation without possible negative effects on cell multiplication and biomass accumulation is desirable. A previous study at our laboratory identified that the CrGPDH3 gene from C. reinhardtii was inducible under NaCl treatments. In this study, we cloned and characterized a 3012 bp sequence upstream of the start codon of the CrGPDH3 gene, including the 285 bp 5' untranslated region. This region was identified as the full-length promoter and named PromA (- 2727 to + 285). Deletion analysis of PromA using GUSPlus as a reporter gene enabled us to identify PromC (- 653 to + 285) as the core promoter, displaying basal expression. A region named RIA1 (- 2727 to - 1672) was suggested to contain the NaCl response elements. Moreover, deletion analysis of RIA1 enabled us to identify a region of 577 bp named RIA3 (- 2727 to - 2150) that, when cloned upstream of PromC, was able to drive the expression of GUSPlus in response to 5 and 100 mM NaCl, and 100 mM KCl, similar to the native CrGPDH3 promoter. These results expand our understanding of the transcriptional mechanism of CrGPDH3 and clearly show that CrGPDH3 promoter and its chimeric forms are highly salt-inducible and can be used as inducible promoters for the overexpression of transgenes in C. reinhardtii.

Temporal distribution and genetic variants in influenza A(H1N1)pdm09 virus circulating in Mexico, seasons 2012 and 2013.

The 2012 and 2013 annual influenza epidemics in Mexico were characterized by presenting different seasonal patterns. In 2012 the A(H1N1)pdm09 virus caused a high incidence of influenza infections after a two-year period of low circulation; whereas the 2013 epidemic presented circulation of the A(H1N1)pdm09 virus throughout the year. We have characterized the molecular composition of the Hemagglutinin (HA) and Neuraminidase (NA) genes of the A(H1N1)pdm09 virus from both epidemic seasons, emphasizing the genetic characteristics of viruses isolated from Yucatan in Southern Mexico. The molecular analysis of viruses from the 2012 revealed that all viruses from Mexico were predominantly grouped in clade 7. Strikingly, the molecular characterization of viruses from 2013 revealed that viruses circulating in Yucatan were genetically different to viruses from other regions of Mexico. In fact, we identified the occurrence of two genetic variants containing relevant mutations at both the HA and NA surface antigens. There was a difference on the temporal circulation of each genetic variant, viruses containing the mutations HA-A141T / NA-N341S were detected in May, June and July; whereas viruses containing the mutations HA-S162I / NA-L206S circulated in August and September. We discuss the significance of these novel genetic changes.

A new virus-induced gene silencing vector based on Euphorbia mosaic virus-Yucatan peninsula for NPR1 silencing in Nicotiana benthamiana and Capsicum annuum var. Anaheim.

Virus-induced gene silencing is based on the sequence-specific degradation of RNA. Here, a gene silencing vector derived from EuMV-YP, named pEuMV-YP:ΔAV1, was used to silence ChlI and NPR1 genes in Nicotiana benthamiana. The silencing of the ChlI transcripts was efficient in the stems, petioles and leaves as reflected in tissue bleaching and reduced transcript levels. The silencing was stable, reaching the flowers and fruits, and was observed throughout the life cycle of the plants. Additionally, the silencing of the NPR1 gene was efficient in both N. benthamiana and Capsicum annuum. After silencing, the plants' viral symptoms increased to levels similar to those seen in wild-type plants. These results suggest that NPR1 plays a role in the compatible interactions of EuMV-YP N. benthamiana and EuMV-C. annum var. anaheim.

Isolation of Peptide aptamers to target protein function.

Peptide aptamers are small recombinant proteins typically inserted into a supportive protein scaffold. These short peptide domains can bind to their target proteins with high specificity and affinity, often resulting in an altered target protein. We describe high-throughput protocols that facilitate the selection and characterization of peptide aptamers from yeast dihybrid libraries. These protocols include the preparation and evaluation of the bait fusion and the peptide aptamer screen. They also include confirmation of interaction specificity as well as isolation and sequencing of peptide inserts. Once the amino acid sequence is determined, we describe a protocol for aligning and comparing short peptide sequences and assessing the statistical significance of the alignments.

Structural relationships between diverse cis-acting elements are critical for the functional properties of a rbcS minimal light regulatory unit.

Light-regulation of photosynthesis-associated nuclear genes is mediated by multipartite cis-regulatory units located in their promoter regions. The combination, spacing, and relative orientation of transcription factor binding sites in these units influences the assembly of specific multiprotein complexes that control gene expression. In this work, the functional architecture of the conserved modular array 5 (CMA5), a previously characterized minimal light-regulatory unit of rbcS gene promoters, has been analysed. With the aim of defining the sequences of CMA5 that, besides the I- and G-box elements, are essential for CMA5 responsiveness to light and chloroplast-derived signals, a series of mutations affecting the sequences flanking these elements was performed. It was found that an I-box associated module, named IbAM5, is essential for CMA5 functionality and is able to bind nuclear proteins in vitro. The spacing requirements of the I- and G-box elements in achieving adequate combinatorial interaction were also studied as well as the effect of interchanging the relative position of these elements in the native rbcS promoter arrangement. The results show that helical phasing and distance between the I- and G-box motifs are critical to determine the functionality and transcriptional strength of CMA5. Furthermore, it is shown that the relative position of the IbAM5/I-box composite element and the G-box element is not critical for the enhancer activity of CMA5, as long as the proper distance between them is maintained. Taken together, these results suggest that the light-responsive, plastid-dependent activity of CMA5 requires the synergistic interaction of several DNA-binding transcription factors assembled in a higher-order nucleoprotein complex.

Peptide aptamers that bind to a geminivirus replication protein interfere with viral replication in plant cells.

The AL1 protein of tomato golden mosaic virus (TGMV), a member of the geminivirus family, is essential for viral replication in plants. Its N terminus contains three conserved motifs that mediate origin recognition and DNA cleavage during the initiation of rolling-circle replication. We used the N-terminal domain of TGMV AL1 as bait in a yeast two-hybrid screen of a random peptide aptamer library constrained in the active site of the thioredoxin A (TrxA) gene. The screen selected 88 TrxA peptides that also bind to the full-length TGMV AL1 protein. Plant expression cassettes corresponding to the TrxA peptides and a TGMV A replicon encoding AL1 were cotransfected into tobacco protoplasts, and viral DNA replication was monitored by semiquantitative PCR. In these assays, 31 TrxA peptides negatively impacted TGMV DNA accumulation, reducing viral DNA levels to 13 to 64% of those of the wild type. All of the interfering aptamers also bound to the AL1 protein of cabbage leaf curl virus. A comparison of the 20-mer peptides revealed that their sequences are not random. The alignments detected seven potential binding motifs, five of which are more highly represented among the interfering peptides. One motif was present in 18 peptides, suggesting that these peptides interact with a hot spot in the AL1 N terminus. The peptide aptamers characterized in these studies represent new tools for studying AL1 function and can serve as the basis for the development of crops with broad-based resistance to single-stranded DNA viruses.

A novel motif in geminivirus replication proteins interacts with the plant retinoblastoma-related protein.

The geminivirus replication factor AL1 interacts with the plant retinoblastoma-related protein (pRBR) to modulate host gene expression. The AL1 protein of tomato golden mosaic virus (TGMV) binds to pRBR through an 80-amino-acid region that contains two highly predicted alpha-helices designated 3 and 4. Earlier studies suggested that the helix 4 motif, whose amino acid sequence is strongly conserved across geminivirus replication proteins, plays a role in pRBR binding. We generated a series of alanine substitutions across helix 4 of TGMV AL1 and examined their impact on pRBR binding using yeast two-hybrid assays. These experiments showed that several helix 4 residues are essential for efficient pRBR binding, with a critical residue being a leucine at position 148 in the middle of the motif. Various amino acid substitutions at leucine-148 indicated that both structural and side chain components contribute to pRBR binding. The replication proteins of the geminiviruses tomato yellow leaf curl virus and cabbage leaf curl virus (CaLCuV) also bound to pRBR in yeast dihybrid assays. Mutation of the leucine residue in helix 4 of CaLCuV AL1 reduced binding. Together, these results suggest that helix 4 and the conserved leucine residue are part of a pRBR-binding interface in begomovirus replication proteins.

Functional properties and regulatory complexity of a minimal RBCS light-responsive unit activated by phytochrome, cryptochrome, and plastid signals.

Light-inducible promoters are able to respond to a wide spectrum of light through multiple photoreceptor systems. Several cis-acting elements have been identified as components of light-responsive promoter elements; however, none of these regulatory elements by itself appears to be sufficient to confer light responsiveness; rather, the combination of at least two elements seems to be required. Using phylogenetic structural analysis, we have identified conserved DNA modular arrays (CMAs) associated with light-responsive promoter regions that have been conserved throughout the evolutionary radiation of angiosperms. Here, we report the functional characterization of CMA5, a native 52-bp fragment of the Nicotiana plumbaginifolia rbcS 8B promoter, which contains an I- and a G-box cis-element. CMA5 behaves as a light-responsive minimal unit capable of activating a heterologous minimal promoter in a phytochrome-, cryptochrome-, and plastid-dependent manner. We also show that CMA5 light induction requires HY5 and that downstream negative regulators COP (constitutive photomorphogenic)/DET (de-etiolated) regulate its activity. Our results show that the simplest light-responsive promoter element from photosynthesis-associated genes described to date is the common target for different signals involved in light regulation. The possible mechanism involved in light-transcriptional regulation and tissue specificity of combinatorial elements units is discussed.