The
gene cluster encodes protein receptors that are potentially able to recognize microbial products and activate signaling pathways that lead to plant cell immunity. Although there are several
...homologs in the tomato genome, only one of them, named
, has been extensively studied due to its functionality against a wide range of (thrips-transmitted) orthotospoviruses. The
gene is a dominant resistance gene originally from a wild Peruvian tomato that has been used in tomato breeding programs aiming to develop cultivars with resistance to these viruses. Here, we provide an overview starting from the first reports of Sw-5 resistance, positional cloning and the sequencing of the
gene cluster from resistant tomatoes and the validation of Sw-5b as the functional protein that triggers resistance against orthotospoviruses. Moreover, molecular details of this plant-virus interaction are also described, especially concerning the roles of Sw-5b domains in the sensing of orthotospoviruses and in the signaling cascade leading to resistance and hypersensitive response.
The COVID-19 pandemic has demanded a range of biotechnological products for detection of SARS-CoV-2 variants and evaluation of human seroconversion after infection or vaccination. In this work, we ...describe an easy pipeline for expression of SARS-CoV-2 nucleocapsid (N) protein in insect cells followed by its purification via affinity chromatography. The N gene was cloned into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) via transposition and the resulting recombinant baculovirus was used for infection of lepidopteran Sf9 cells adapted to high-density suspension. Using Tris−HCl pH 8.0 buffer as mobile phase and eluting bound proteins with 175 mM imidazole as part of a three-step gradient, an average of 1 mg N protein could be purified from each 50 mg of total protein from clarified supernatant. Such protein amount allows the manufacturing of serological tests and the development of basic studies on cellular responses to SARS-CoV-2.
Although RNA viruses have high mutation rates, host cells and organisms work as selective environments, maintaining the viability of virus populations by eliminating deleterious genotypes. In serial ...passages of RNA viruses in a single cell line, most of these selective bottlenecks are absent, with no virus circulation and replication in different tissues or host alternation. In this work,
Aag-2 cells were accidentally infected with Chikungunya virus (CHIKV) and Mayaro virus (MAYV). After numerous passages to achieve infection persistency, the infectivity of these viruses was evaluated in
C6/36 cells, African green monkey Vero cells and primary-cultured human fibroblasts. While these CHIKV and MAYV isolates were still infectious to mosquito cells, they lost their ability to infect mammalian cells. After genome sequencing, it was observed that CHIKV accumulated many nonsynonymous mutations and a significant deletion in the coding sequence of the hypervariable domain in the
gene. Since MAYV showed very low titres, it was not sequenced successfully. Persistently infected Aag-2 cells also accumulated high loads of short and recombinant CHIKV RNAs, which seemed to have been originated from virus-derived DNAs. In conclusion, the genome of this CHIKV isolate could guide mutagenesis strategies for the production of attenuated or non-infectious (to mammals) CHIKV vaccine candidates. Our results also reinforce that a paradox is expected during passages of cells persistently infected by RNA viruses: more loosening for the development of more diverse virus genotypes and more pressure for virus specialization to this constant cellular environment.
Pepper mild mottle virus (PMMoV), a tobamovirus, has been reported worldwide infecting, especially, pepper plants. In this study, seven new PMMoV isolates were identified in symptomatic pepper plants ...collected in four departments of Peru. The majority of these isolates induced severe symptoms, including mottle, in susceptible pepper plants. Inoculation on pepper plants carrying four known functional
L
resistance alleles (
L
1
,
L
2
,
L
3
, and
L
4
) revealed that PMMoV isolates from Peru belong to the P
1,2
pathotype. Therefore, pepper species or cultivars with
L
3
and
L
4
alleles displayed hypersensitive response after mechanical inoculation and were still resistant to PMMoV isolates circulating in Peru. Infection by PMMoV was monitored by serology (DAS-ELISA) and RT-PCR with degenerate primers. Virus genes were used to build a phylogenetic tree that showed diversity accumulation among the PMMoV isolates from Peru.
Orthotospoviruses (genus
Orthotospovirus
, family
Tospoviridae
) are amongst the most devastating plant viruses worldwide, causing severe damage to many economically important vegetable crops, such ...as tomato and sweet pepper. Monitoring virus populations is an important step for estimating virus damage and epidemiology, and gaining insights into the adaptation processes undergone by orthotospoviruses. Here, we studied the orthotospovirus populations infecting vegetable crops in Brazil and the Dominican Republic, including species diversity, genome comparison and phylogenetic analyses. Comparisons of virus populations showed that in Brazil, which is considered a center of orthotospovirus diversity, groundnut rinspot virus (GRSV) is prevalent, infecting 41% of the plants, whereas tomato spotted wilt virus (TSWV) and tomato chlorotic spot virus (TCSV) were present in 4% and 9% of the samples, respectively. In the Dominican Republic, which can be considered an environment with low orthotospovirus diversity, 55% of the samples were infected with TSWV, 11% showed TCSV infection and no GRSV was detected. The occurrence of mixed infection was low in Brazil, at only 5%, but no mixed infection was detected in the Dominican Republic. The low rates of mixed infections may prevent the emergence of genomes resulting from reassortment. Indeed, no reassortant viruses were detected in either country, except for TCSV, recently proposed as representing a reassortant orthotospovirus species.
Objetivou-se com este estudo avaliar os efeitos da aplicação de solução nutritiva com concentrações crescentes de zinco (Zn) no desenvolvimento inicial e no teor de pigmentos de plantas de feijão ...cultivadas em condições de casa de vegetação. O experimento foi desenvolvido no Laboratório de Nutrição de Plantas (LNP) e em casa de vegetação pertencentes ao Departamento de Botânica da Universidade Federal de Pelotas (UFPel). A semeadura foi realizada em vasos preenchidos com areia média lavada. Sete dias após a emergência das plântulas foi aplicada, com intervalos de dois dias, solução nutritiva com quatro concentrações crescentes de Zn (2, 50, 75 e 100 µM), dispostas em um delineamento experimental inteiramente casualizado com quatro repetições, sendo cada uma delas composta por seis plantas. Aos 45 dias após a emergência das plântulas, avaliaram-se as seguintes variáveis: altura das plantas, diâmetro do caule, número de folhas, área foliar e comprimento de raiz, além do índice de pigmentos fotossintéticos. Com o aumento das concentrações de Zn verificou-se decréscimo significativo para as variáveis diâmetro do caule e comprimento de raiz, sugerindo que maiores concentrações de Zn podem influenciar negativamente no desenvolvimento das plantas de feijão. Os índices de clorofila e flavonoides apresentaram elevação com o aumento das doses de Zn. Esse efeito possivelmente está relacionado a uma reação estratégica da espécie, que visa minimizar o efeito tóxico das concentrações crescentes de Zn no desenvolvimento das plantas.Palavras-chave: Feijão. Micronutriente. Pigmentos foliares. Toxicidade.
Tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV) share several genetic and biological traits. Both of them belong to the genus
(family
), which is composed by viruses with ...tripartite RNA genome that infect plants and are transmitted by thrips (order Thysanoptera). Previous studies have suggested several reassortment events between these two viruses, and some speculated that they may share one of their genomic segments. To better understand the intimate evolutionary history of these two viruses, we sequenced the genomes of the first TCSV and GRSV isolates ever reported. Our analyses show that TCSV and GRSV isolates indeed share one of their genomic segments, suggesting that one of those viruses may have emerged upon a reassortment event. Based on a series of phylogenetic and nucleotide diversity analyses, we conclude that the parental genotype of the M segment of TCSV was either eliminated due to a reassortment with GRSV or it still remains to be identified.
Mayaro virus (MAYV) is responsible for a mosquito-borne tropical disease with clinical symptoms similar to dengue or chikungunya virus fevers. In addition to the recent territorial expansion of MAYV, ...this virus may be responsible for an increasing number of outbreaks. Currently, no vaccine is available. Aedes aegypti is promiscuous in its viral transmission and thus an interesting model to understand MAYV-vector interactions. While the life-cycle of MAYV is known, the mechanisms by which this arbovirus affects mosquito host cells are not clearly understood.
After defining the best conditions for cell culture harvesting using the highest virus titer, Ae. aegypti Aag-2 cells were infected with a Brazilian MAYV isolate at a MOI of 1 in order to perform a comparative proteomic analysis of MAYV-infected Aag-2 cells by using a label-free semi-quantitative bottom-up proteomic analysis. Time-course analyses were performed at 12 and 48 h post-infection (hpi). After spectrum alignment between the triplicates of each time point and changes of the relative abundance level calculation, the identified proteins were annotated and using Gene Ontology database and protein pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes.
After three reproducible biological replicates, the total proteome analysis allowed for the identification of 5330 peptides and the mapping of 459, 376 and 251 protein groups, at time 0, 12 hpi and 48 hpi, respectively. A total of 161 mosquito proteins were found to be differentially abundant during the time-course, mostly related to host cell processes, including redox metabolism, translation, energy metabolism, and host cell defense. MAYV infection also increased host protein expression implicated in viral replication.
To our knowledge, this first proteomic time-course analysis of MAYV-infected mosquito cells sheds light on the molecular basis of the viral infection process and host cell response during the first 48 hpi. Our data highlight several mosquito proteins modulated by the virus, revealing that MAYV manipulates mosquito cell metabolism for its propagation.
Tospoviruses (Genus Tospovirus, Family Bunyaviridae) are phytopathogens responsible for significant worldwide crop losses. They have a tripartite negative and ambisense RNA genome segments, termed S ...(Small), M (Medium) and L (Large) RNA. The vector-transmission is mediated by thrips in a circulative-propagative manner. For new tospovirus species acceptance, several analyses are needed, e.g., the determination of the viral protein sequences for enlightenment of their evolutionary history.
Biological (host range and symptomatology), serological, and molecular (S and M RNA sequencing and evolutionary studies) experiments were performed to characterize and differentiate a new tospovirus species, Bean necrotic mosaic virus (BeNMV), which naturally infects common beans in Brazil. Based upon the results, BeNMV can be classified as a novel species and, together with Soybean vein necrosis-associated virus (SVNaV), they represent members of a new evolutionary lineage within the genus Tospovirus. CONCLUSION/SIGNIFICANCES: Taken together, these evidences suggest that two divergent lineages of tospoviruses are circulating in the American continent and, based on the main clades diversity (American and Eurasian lineages), new tospovirus species related to the BeNMV-SVNaV clade remain to be discovered. This possible greater diversity of tospoviruses may be reflected in a higher number of crops as natural hosts, increasing the economic impact on agriculture. This idea also is supported since BeNMV and SVNaV were discovered naturally infecting atypical hosts (common bean and soybean, respectively), indicating, in this case, a preference for leguminous species. Further studies, for instance a survey focusing on crops, specifically of leguminous plants, may reveal a greater tospovirus diversity not only in the Americas (where both viruses were reported), but throughout the world.
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