Biology subjects

Velasco, L.

Publications and source records attributed to Velasco, L..

6 recordsLinked to original sources

bioRxiv2026

Fine mapping and genomic analyses reveal a tandem kinase-pseudokinase candidate underlying OrDeb2-mediated resistance to Orobanche cumana in sunflower

Sunflower broomrape (Orobanche cumana Wallr.) is a holoparasitic weed that parasitizes sunflower (Helianthus annuus L.) and severely constrains yield. The OrDeb2 gene confers resistance to broomrape race G in the sunflower line DEB2. It has been previously located within a 1.38-Mbp physical region in the H. annuus XRQ reference genome that contains a cluster of receptor-like proteins and receptor-like kinases (RLKs). In this study, an OrDeb2 candidate has been proposed though high-resolution genetic and physical mapping, multi-allelic comparisons across resistant and susceptible lines, and exclusion of other potential candidates based on their function and genetic features. OrDeb2 was delimited to a 219.4-kbp region in the DEB2 genome assembly that contained six annotated protein-coding genes belonging to two groups: three small heat shock proteins (HSP20) and three RLKs. Given that the only sunflower broomrape resistance gene cloned to date encodes an RLK, the key role of RLKs in resistance to biotic stresses in plants, and the similar genetic features between RLK-type resistance genes and OrDeb2 as major dominant genes in gene-for-gene interactions, RLKs in the interval emerged as the most plausible candidates. Within them, the RLK-2 gene encoding a root-expressed full-length kinase-pseudokinase protein belonging to the tandem kinase proteins (TKPs) family was prioritized, because the other two RLKs corresponded to a truncated TKP with a stop codon within the kinase domain. RLK-2 also carried sequence variants unique to DEB2 relative to multiple susceptible genotypes. The results of this research reveal the potential role of TKP genes as new players in resistance to parasitic plants and provide a framework to dissect the genetic dialog between O. cumana and sunflower in gene-for-gene interactions.

plant biology↗

bioRxiv2024

Topical application of carbon dots and mesoporous silica nanoparticle-derived dsRNA-nanocomposites for the control of beet curly top virus and turnip mosaic virus

The management of emerging phytoviruses in current agriculture confronts many challenges, including the appearance of new strains or the arrival of new species, requiring a multidisciplinary approach. The usual methods for the control of these pathogens are based on management practices, vector control, seed control, etc., and the introgression of genetic resistance in cultivars. But the development of resistances by classical genetic methods is a costly and time-consuming process. A promising alternative is the control of these pathogens by means of the so-called SIGS (Spray-Induced Gene Silencing) that consists of topical treatments with specific molecules derived from the viruses as double-stranded RNA (dsRNA), triggering the plant defense mechanisms. In this work, we show the control of two viruses, the potyvirus turnip mosaic virus (TuMV) and the curtovirus beet curly top virus (BCTV) by dsRNA nanocomposites with carbon dots or mesoporous silica nanoparticles in Nicotiana benthamiana. In the case of TuMV, the disease was significantly reduced in terms of plant photosynthetic capacity and viral titers, in the dsRNA treated plants. Moreover, when the treatments were carried out with dsRNAs as nanocomposites, the differences were even more noticeable with respect to untreated inoculated plants. In the case of BCTV, a significant delay in symptoms appearance was observed after the treatments with the dsRNA nanocomposites, but not with the naked dsRNAs. Viral titers were reduced either with the naked or the nanoparticle-delivered dsRNAs. The increased efficiency of dsRNA for virus control when supplied with the nanoparticles can be related with the enhanced dsRNA delivery reported in this work.

plant biology↗

bioRxiv2024

Genome-wide screen overexpressing mycobacteriophage Amelie genes identifies multiple inhibitors of mycobacterial growth

The genome sequences of thousands of bacteriophages have been determined and functions for many of the encoded genes have been assigned based on homology to characterized sequences. However, functions have not been assigned to more than two-thirds of the identified phage genes as they have no recognizable sequence features. Recent genome-wide overexpression screens have begun to identify bacteriophage genes that encode proteins that reduce or inhibit bacterial growth. This study describes the construction of a plasmid-based overexpression library of 76 genes encoded by Cluster K1 mycobacteriophage Amelie, which is genetically similar to Cluster K phages Waterfoul and Hammy recently described in similar screens and closely related to phages that infect clinically important mycobacteria. 26 out of the 76 genes evaluated in our screen, encompassing 34% of the genome, reduced growth of the host bacterium Mycobacterium smegmatis to various degrees. More than one-third of these 26 toxic genes have no known function, and 10 of the 26 genes almost completely abolished host growth upon overexpression. Notably, while several of the toxic genes identified in Amelie shared homologs with other Cluster K phages recently screened, this study uncovered eight previously unknown gene families that exhibit cytotoxic properties, thereby broadening the repertoire of known phage-encoded growth inhibitors. This work, carried out under the HHMI-supported SEA-GENES project (Science Education Alliance Gene-function Exploration by a Network of Emerging Scientists), underscores the importance of comprehensive overexpression screens in elucidating genome-wide patterns of phage gene function and novel interactions between phages and their hosts.

microbiology↗

bioRxiv2022

Carbon dots boost dsRNA delivery in plants and increase local and systemic siRNA production

Carbon dots (CDs) have been proposed for nucleic acid delivery in many biological applications. In this work we have obtained carbon dots by a hydrothermal synthesis method for developing nanocomposites with dsRNA. These CDs were produced using glucose or saccharose as the nucleation source and passivated with branched polyethyleneimines for conferring positive charges. Hydrodynamic analyses and transmission electron microscopy TEM showed that they sized on average 4 and 5 nm, depending on the sugar. The CDs were fluorescent and showed a peak at 468 nm when excited with UV light. Physicochemical characteristics of their surfaces were revealed by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FT-IR). The {zeta} potential determined that both types of CDs had positive charges as well as good electrophoretic mobility and conductivity. Coating of the CDs to dsRNA was efficient but did not protect from nucleases. DsRNA naked or coated with the CDs were delivered to leaves of cucumber plants by spraying at 2.5 bar. Quantitation of the dsRNA that entered the leaves showed that when coated with the CDs, 50-fold more dsRNA was detected than when naked dsRNA was applied. Moreover, specific siRNAs derived from the sprayed dsRNAs were 130 times more abundant when the dsRNA was coated with the CDs. Systemic dsRNAs were determined in distal leaves showing a dramatic increase in concentration when delivered as a nanocomposite. Similarly, systemic siRNAs were significantly more abundant in distal leaves when spraying with the CD-dsRNA nanocomposite. Furthermore, FITC-labeled dsRNA was shown to accumulate in the apoplast and increase its entry in the plant when coated with CDs. These results indicate that CDs obtained by hydrothermal synthesis are suitable for dsRNA foliar delivery in RNAi plant applications and provide insight into CD-dsRNA translocations.

plant biology↗

bioRxiv2022

Exogenous application of dsRNA for the control of viruses in cucurbits

The recurrent emergence of virus diseases in intensive horticultural crops requires alternative control strategies. Topical application of dsRNA molecules homologous to pathogens has been proposed as a tool for plant virus control. These dsRNAs induce the silencing mechanism (RNAi) that degrades homologous dsRNAs. Cucumber green mottle mosaic virus (CGMMV) represents a serious threat to cucurbit crops. Since genetic resistance to the virus is not yet available in commercial varieties, we aimed to control this virus by RNAi. For this purpose, we obtained constructions both for expressing dsRNA in bacteria to treat cucumber plants by topical application and for agroinoculation in experiments done in the growth chamber. Besides, greenhouse tests were performed in spring and in summer when plants were challenged with the virus and differences in several parameters were investigated, including severity of symptoms, dry weight, total height, virus accumulation and virus-derived small interfering RNAs (vsiRNAs). Spraying of plants with dsRNA reduced significatively CGMMV symptoms in the plants in growth chamber tests. Agroinfiltration experiments done under identical conditions were also effective in limiting the progress of CGMMV disease. In the greenhouse assay performed in spring, symptoms were significatively reduced in dsRNA-sprayed plants and the development of the plants improved with respect to non-treated plants. Virus titters and vsiRNAs were clearly reduced in dsRNA-treated plants. The effect of protection of the dsRNA was less evident in the greenhouse assay carried out in summer. Besides, we investigated the mobility of long (ds)RNA derived from spraying or agroinfiltrated dsRNA and found that it could be detected in local, close distal and far distal points from the site of application. VsiRNAs were also detected in local and distal points and the differences in accumulation were compared. In parallel, we investigated the capacity of dsRNAs derived from genes of tomato leaf curl New Delhi virus (ToLCNDV), another economically important virus in cucurbits, to limit the disease in zucchini, both by agroinfiltration or direct spraying, but found no protection effect. In view of the results, topical application of dsRNAs is postulated as a promising strategy for CGMMV control in cucumber.

plant biology↗

bioRxiv2018

First natural crossover recombination of intact ORFs between two distinct species of the family Closteroviridae

Lettuce chlorosis virus-SP (LCV-SP) (family Closteroviridae, genus Crinivirus), is a new strain of LCV which is able to infect green bean plants and incapable of infecting lettuce crops. In the present study, high throughput and Sanger sequencing of RNA was used to obtain the LCV-SP full-length sequence. The LCV-SP genome comprises 8825 nt and 8672 nt equivalent with RNA1 and RNA2 respectively. RNA1 of LCV-SP contains four ORFs, the proteins encoded by the ORF1a and ORF1b are closely related to LCV RNA1 from California (FJ380118) whereas the 3{acute} end encodes proteins which share high amino acid sequence identity with RNA1 of BnYDV (EU191904). The genomic sequence of RNA2 consists of 8 ORFs, instead of 10 ORFs contained in LCV-California isolate. The distribution of vsiRNA (virus-derived small interfering RNA) along the LCV-SP genome suggested the presence of subgenomic RNAs corresponding with HSP70, P6.4 and P60. Results of the analysis using RDP4 and Simplot programs are the proof of the evidence that LCV-SP is the first recombinant of the family Closteroviridae by crossover recombination of intact ORFs, being the LCV RNA1 (FJ380118) and BnYDV RNA1 (EU191904) the origin of the new LCV strain. Genetic diversity values of virus isolates in the recombinant region obtained after sampling LCV-SP infected green bean between 2011 and 2017 might suggest that the recombinant virus event occurred in the area before this period. The presence of LCV-SP shows the role of recombination as a driving force of evolution within the genus Crinivirus, a globally distributed, emergent genus.

plant biology↗