Antitumor and immunostimulatory activities of a genotype V recombinantattenuated veterinary Newcastle disease virus vaccine

"Antitumor conventional treatments including chemo/radiotherapy result in several side effects and non-specificity. Therapies including the use of oncolytic viruses, particularly the Newcastle disease virus (NDV), have emerged as an attractive alternative due to their capacity to kill cancer cells directly or through stimulation of the immune system. In the present study, a commercial vaccine composed of a recombinant attenuated NDV strain P05 (rNDV-P05) was assessed for antitumor and immunostimulatory activity. Firstly, hemagglutination activity was evaluated at different pH and temperature conditions. Then, cancer cell lines and peripheral blood mononuclear cells (PBMC) were co-cultured with or without rNDV-P05 and cytoplasmic nucleosomes were measured by enzyme-linked immunosorbent assay (ELISA) as an apoptosis indicator. Antitumor cytokines produced by PBMC in response to the virus were analyzed by ELISA and reverse transcription quantitative polymerase chain reaction. Characterization ofrNDV-P05 indicates that the virus is slightly sensible to acid and basic pH, and stable at temperatures no greater than 42 degrees C. The majority of cell lines developed apoptosis in co-culture with rNDV-P05 in a dose-time dependent manner. The highest level of HeLa, HCC1954 and HepG2 cell apoptosis was at 48 h/50 hemagglutination units (HU), and HL-60 was 24 h/50 HU.A549 cell line and PBMC did not show sensitivity to apoptosis by the virus. PBMC from healthy donors stimulated with the rNDV-P05 increased significantly the levels of interferon (IFN)-alpha, IFN-gamma, tumor necrosis factor (TNF)-alpha and soluble TNF-related apoptosis-inducing I igand in culture supernatants, as well as their mRNA expression. These results demonstrate that the pro-apoptotic effect ofrNDV-P05 and its magnitude is specific to particular tumor cell lines and is not induced on PBMC; and the virusstimulates the expression of several key antitumor cytokines. This study promotes the use of rNDV-P05 in an alternate application of different viral strains during virotherapy with NDV.

Evaluation of a SUMO E2 conjugating enzyme involved in resistance to Clavibacter michiganensis subsp. michiganensis in Solanum peruvianum, through a tomato mottle virus VIGS assay

"Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker of tomato. Currently, no Solanum lycopersicum resistant varieties are commercially available, but some degree of Cmm resistance has been identified in Solanum peruvianurn. Previous research showed up regulation of a SUMO E2 conjugating enzyme (SCEI) transcript in S. peruvianum compared to S. lycopersicum following infection with Cmm. In order to test the role of SCEI in resistance to Cmm, a fragment of SCEI from S. peruvianum was cloned into a novel virus induced gene silencing (VIGS) vector based on the geminivirus, Tomato Mottle Virus (ToMoV). Using biolistic inoculation, the ToMoV-based VIGS vector was shown to be effective in S. peruvianum by silencing the magnesium chelatase gene, resulting in leaf bleaching. VIGS with the ToMoV_SCEI construct resulted in 61% silencing of SCEI in leaves of S. peruvianum as determined by quantitative RT-PCR. The SCEI -silenced plants showed unilateral wilting (15 dpi) and subsequent death (20 dpi) of the entire plant after Cmm inoculation, whereas the empty vector-treated plants only showed wilting in the Cmm-inoculated leaf. The SCEI-silenced plants showed higher Cmm colonization and an average of 4.5 times more damaged tissue compared to the empty vector control plants. SCEI appears to play an important role in the innate immunity of S. peruvianum against Cmm, perhaps through the regulation of transcription factors, leading to expression of proteins involved in salicylic acid dependent defense responses.

Estudio funcional de genes de defensa contra el chancro bacteriano, mediante vectores virales en especies silvestres relacionadas al tomate

"El tomate, Solanum lycopersicum, representa uno de los cultivares de mayor consumo a nivel mundial, su producción se ha visto afectada por diversos patógenos, siendo el más importante Clavibacter michiganensis subsp. michiganensis (Cmm). La enfermedad causada por Cmm se conoce como chancro bacteriano, contra la cual no se tiene tratamiento efectivo actualmente; sin embargo, se han identificado especies silvestres relacionadas al tomate, que son resistentes a Cmm, estas representan una fuente de genes que podrían transferirse a la especie susceptible. En este trabajo se analizó la función de genes potencialmente involucrados en la resistencia a Cmm mediante su silenciamiento inducido por virus (VIGS) y el subsecuente reto con el patógeno. Se analizaron dos genes en particular, uno que codifica a la enzima de conjugación SUMO E2 (SCEI), y otro que codifica para un transportador de la familia ABC, que confiere resistencia a drogas pleiotrópicas (PDRI). Se diseñó un vector VIGS con base en el Virus Moteado del Tomate (ToMoV), para el análisis funcional. El silenciamiento del gen SCEI en S. peruvianum no produjo ningún fenotipo aberrante, lo que permitió su análisis subsecuente en experimentos de inoculación con Cmm. Las plantas silenciadas y retadas con Cmm mostraron un incremento en la susceptibilidad a este patógeno, lo que nos permite sugerir la participación de SCEI en el mecanismo de defensa. Por otra parte, el silenciamiento del gen PDRI en S. habrochaites realizado mediante un vector TRV, no alteró significativamente la respuesta de las plantas al reto con Cmm, lo que sugiere que este gen no juega un papel crucial en la resistencia a este patógeno o el silenciamiento no fue eficiente. Sin embargo, el silenciamiento del mismo gen con el vector derivado de ToMoV resultó en un ligero aumento en la susceptibilidad a Cmm, aunque el nivel de daño no fue significativo estadísticamente. La sobreexpresión de los genes SCEI y PDRI en plantas susceptibles puede ser una estrategia de control del cáncer bacteriano.""Tomato, S. lycopersicum, is one of the crops most consumed worldwide, and its production is affected by several pathogens, among these the most important is the bacteria Clavibacter michiganensis subsp. michiganensis (Cmm). The disease caused by Cmm is known as bacterial canker and currently there is no effective treatment, however, there are reports where wild tomato relatives show some resistance to Cmm, thus representing a source of genes that could be transferred to susceptible species. In this study, were analyzed the function of genes potentially involved in the resistance to Cmm by virus induced gene silencing (VIGS) and the subsequent challenge with the pathogen. Two genes in particular where analyzed, the first one encodes the SUMO E2 conjugating enzyme (SCEI); and the second encodes an ABC family transporter that confers, pleiotropic drug resistance (PDRI). A VIGS vector was designed based on the Tomato Mottle Virus (ToMoV). SCEI gene silencing in S. peruvianum did not showed any aberrant phenotype, which allowed its subsequent analysis of inoculation experiments with Cmm. Plants that were silenced for SCEI gene and then challenged with Cmm showed increased susceptibility to this pathogen, suggesting its role in the defense mechanism against Cmm. On the other hand, PDRI gene silencing in S. habrochaites performed by TRV vector, did not alter significantly the response of the plants to the challenge with Cmm, which suggests either that this gene is not implicated in the resistance to this pathogen or the silencing with TRV was not efficient. However, the silencing of the same gene with the vector derived from ToMoV resulted in a slight increase on the susceptibility to Cmm; however, the level of damage was not statistically significant. Overexpression of the SCEI and PDRI genes in susceptible plants can be a control strategy against bacterial canker.

Response Surface Methodology for Optimization of Multiplex-PCR Protocols for Detection of TYLCV, TSWV and <i>Fol</i> Molecular Markers: Analytical Performance Evaluation

Molecular markers linked to disease resistance genes which affect economically important crops are of great interest. In the case of tomato, a major focus on resistance breeding to multiple fungal and viral pathogens such as Tomato yellow leaf curl virus (TYLCV), Tomato spotted wilt virus (TSWV) and Fusarium oxysporum f. sp. lycopersici (Fol), have led to the introgression of several resistance genes; therefore, molecular markers have become important in molecular-assisted selection (MAS) of tomato varieties resistant to those pathogens. However, assays that allow simultaneous evaluation of resistant genotypes, such as multiplex PCR, need to be optimized and evaluated to demonstrate their analytical performance, as many factors can affect them. This work aimed to generate multiplex PCR protocols for the joint detection of the molecular markers associated with pathogen resistance genes in tomato plants that are sensitive, specific and repeatable. For the optimization a central composite design of a response surface methodology (RSM-CCD) was used. For analytical performance evaluation, specificity/selectivity and sensibility (limit of detection and dynamic range) were analyzed. Two protocols were optimized: the first one with a desirability of 1.00, contained two markers (At-2 and P7-43) linked to I- and I-3-resistant genes. The second one with a desirability of 0.99, contained markers (SSR-67, SW5 and P6-25) linked to I-, Sw-5-, and Ty-3-resistant genes. For protocol 1, all the commercial hybrids (7/7) were resistant to Fol, and for protocol 2, two hybrids were resistant to Fol, one to TSWV and one to TYLCV with good analytical performance. In both protocols, the varieties considered susceptible to the pathogens, no-amplicon or susceptible amplicons, were observed. The optimized multiplex PCR protocols showed dynamic ranges from 5.97 up to 161.3 ng DNA. The limit of detection was 17.92 ng and 53.76 ng DNA for protocols 1 and 2, respectively, giving 100% positive results in the test replicates. This method allowed to develop optimized multiplex PCR protocols with few assays which translates into less time and resources, without sacrificing method performance

Evaluation of a SUMO E2 conjugating enzyme involved in resistance to Clavibacter michiganensis subsp. michiganensis in Solanum peruvianum, through a tomato mottle virus VIGS assay

Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker of tomato. Currently, no Solanum lycopersicum resistant varieties are commercially available, but some degree of Cmm resistance has been identified in Solanum peruvianum. Previous research showed up-regulation of a SUMO E2 conjugating enzyme (SCEI) transcript in resistant S. peruvianum compared to susceptible S. lycopersicum following infection by Cmm. In order to test the role of SCEI in resistance to Cmm, a fragment of the gene from S. peruvianum was cloned into a novel virus-induced gene-silencing (VIGS) vector based on the geminivirus Tomato Mottle Virus (ToMoV). Using biolistic inoculation, the ToMoV-based VIGS vector was shown to be effective in S. peruvianum by silencing the magnesium chelatase gene, which resulted in leaf bleaching. The ToMoV_SCEI construct resulted in approx. 61% silencing of SCEI in leaves of S. peruvianum as determined by quantitative RT-PCR. VIGS of SCEI in S. peruvianum resulted in unilateral wilting (15 dpi) and subsequent death (20 dpi) of the entire plant after Cmm inoculation, whereas empty vector-treated plants only showed wilting in the Cmm-inoculated leaf. SCEI-silenced plants also showed higher Cmm colonization with an average of 4.5 times more damaged tissue compared to the empty vector control plants. SCEI appears to play an important role in the innate immunity of S. peruvianum against Cmm, perhaps through the regulation of WRKY transcription factors, which may lead to expression of proteins involved in salicylic acid-dependent defense responses