Viral determinants involves in the differential adaptation of two begomovirus in tomato and Nicotiana benthamiana

Os begomovírus (família Geminiviridae) possuem genoma constituído por um ou dois componentes de DNA de fita simples circular, e infectam plantas dicotiledôneas. Os sintomas induzidos pelo begomovírus Tomato yellow spot virus (ToYSV) em tomateiro e Nicotiana benthamiana são mais severos e surgem mais cedo em comparação aos induzidos pelo Tomato rugose mosaic virus (ToRMV). Além disso, o ToYSV invade o mesofilo em N. benthamiana, enquanto o ToRMV é restrito ao floema nesse hospedeiro (ambos os vírus são restritos ao floema em tomateiro). Essas observações indicam uma adaptação diferencial de cada um desses vírus aos seus hospedeiros. Com o objetivo de mapear os determinantes genéticos virais responsáveis por essa adaptação diferencial, foram construídos três recombinantes com base na troca dos genes MP e NSP e da região BRi entre esses vírus, em diferentes combinações. Os experimentos com os recombinantes foram realizados para determinar o ganho ou a perda das seguintes características: intensidade de sintomas, acúmulo de DNA viral e tropismo de tecido. Os recombinantes recíprocos nos quais os genes MP e NSP foram trocados apresentaram sintomas de intensidade intermediária em comparação aos vírus parentais em ambos os hospedeiros, e o mesmo tropismo de tecido em N. benthamiana. Já o recombinante no qual a região BRi do ToYSV foi inserido no DNA-B do ToRMV foi capaz de infectar o mesofilo em N. benthamiana. Em conjunto, esses resultados sugerem que as proteínas MP e NSP e a região BRi estão envolvidas na adaptação do ToYSV em tomateiro e N. benthamiana, e que a região BRi é o determinante viral de tropismo de tecido em N. benthamiana. Outras proteínas e sequências regulatórias presentes no DNA-A também devem estar envolvidas na adaptação desses vírus, no entanto experimentos adicionais, incluindo a análise do recombinante no qual a região BRi do ToRMV esteja inserida no DNA-B do ToYSV, são necessários para a identificação precisa dessas regiões.Begomoviruses (family Geminiviridae) have a genome composed of a one or two components of circular, single-stranded DNA and infect dicotyledoneous plants. Symptoms induced by the begomovirus Tomato yellow spot virus (ToYSV) in tomato and Nicotiana benthamiana are more severe and appear earlier compared to those induced by Tomato rugose mosaic virus (ToRMV). Furthermore, ToYSV invades the mesophyll in N. benthamiana, whereas ToRMV is phloem-restricted in this host (both viruses are phloem-restricted in tomato). These observations suggest a differential adaptation of each virus to its host. In order to map the viral genetic determinants responsible for this differential adaptation, three recombinants were constructed based on the exchange of the MP and NSP genes and the BRi region between the two viruses, in different combinations. Experiments with the recombinants were performed to determine the gain or loss of the following characteristics: intensity of symptoms, viral DNA accumulation and tissue tropism. Reciprocal recombinants in which the MP and NSP genes were swapped induced symptoms of intermediate intensity compared to the parental viruses in both hosts, and had the same tissue tropism in N. benthamiana. The recombinant in which the ToYSV BRi region was placed in the ToRMV DNA-B background was capable of invading the mesophyll in N. benthamiana. Together, these results indicate that the MP and NSP proteins as well as the BRi region are involved in the adaptation of ToYSV in tomato and N. benthamiana, and that the BRi region is the viral determinant of tissue tropism in N. benthamiana. Other proteins and regulatory sequences present in the DNA-A could also be involved in the adaptation of these viruses, however further experiments, including the analysis of a recombinant in which the BRi region from ToRMV is placed in the ToYSV background, are necessary for their precise identification.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

The dsRNA Virus Papaya Meleira Virus and an ssRNA Virus Are Associated with Papaya Sticky Disease

<div><p>Papaya sticky disease, or “meleira”, is one of the major diseases of papaya in Brazil and Mexico, capable of causing complete crop loss. The causal agent of sticky disease was identified as an isometric virus with a double stranded RNA (dsRNA) genome, named papaya meleira virus (PMeV). In the present study, PMeV dsRNA and a second RNA band of approximately 4.5 kb, both isolated from latex of papaya plants with severe symptoms of sticky disease, were deep-sequenced. The nearly complete sequence obtained for PMeV dsRNA is 8,814 nucleotides long and contains two putative ORFs; the predicted ORF1 and ORF2 display similarity to capsid proteins and RdRp's, respectively, from mycoviruses tentatively classified in the family <i>Totiviridae</i>. The sequence obtained for the second RNA is 4,515 nucleotides long and contains two putative ORFs. The predicted ORFs 1 and 2 display 48% and 73% sequence identity, respectively, with the corresponding proteins of papaya virus Q, an umbravirus recently described infecting papaya in Ecuador. Viral purification in a sucrose gradient allowed separation of particles containing each RNA. Mass spectrometry analysis indicated that both PMeV and the second RNA virus (named papaya meleira virus 2, PMeV2) were encapsidated in particles formed by the protein encoded by PMeV ORF1. The presence of both PMeV and PMeV2 was confirmed in field plants showing typical symptoms of sticky disease. Interestingly, PMeV was detected alone in asymptomatic plants. Together, our results indicate that sticky disease is associated with double infection by PMeV and PMeV2.</p></div

Schematic representation of the genomic organization of papaya umbraviruspapaya meleira virus 2 (PpUVPMeV2).

<p>The viral ssRNA contains two ORFs. ORF1 encodes a hypothetical protein and ORF2 encodes a putative RdRp.</p

Alignment of the RdRp4 domain from PMeV and related viruses.

<p>Alignment of the RdRp4 domain (pfam02123) identified in ORF2 of papaya meleira virus (PMeV) with the corresponding regions of the most closely related totivirid-like viruses (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155240#pone.0155240.s003" target="_blank">S2 Table</a> for full virus names and GenBank access numbers). Sequences were aligned with MUSCLE (Edgar, 2004). The numbers in parenthesis indicate the number of amino acid residues separating individual motifs. Amino acid residues highlighted in black are conserved among all aligned sequences.</p

Genome organization and molecular features of papaya meleira virus (PMeV).

<p>(a) Schematic representation of the PMeV genomic organization. The viral dsRNA contains two ORFs. ORF1 encodes the putative coat protein (CP) and ORF2 encodes a putative RNA-dependent RNA polymerase (RdRp). The position of a putative slippery sequence for -1 translational frameshift is indicated at the 3'-end of ORF1. (b) Predicted pseudoknot structure located downstream of the slippery sequence.</p

Phylogenetic relationship between PpUVPMeV2 and related viruses.

<p>Phylogenetic tree based on an alignment of the deduced amino acid sequences of PpUVPMeV2 and members of the family <i>Tombusviridae</i>, obtained using Bayesian inference. The numbers at the branch nodes indicate Bayesian posterior probabilities. The names of the viruses used in the analysis and their respective GenBank access numbers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155240#pone.0155240.s003" target="_blank">S2 Table</a>.</p

Phylogenetic relationship between PMeV and related viruses.

<p>Phylogenetic tree based on an alignment of deduced amino acid sequences of the RdRp proteins from PMeV and members of the family <i>Totiviridae</i>, obtained using Bayesian inference. The numbers at the branch nodes indicate Bayesian posterior probabilities. The names of the viruses used in the analysis and their respective GenBank access numbers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155240#pone.0155240.s003" target="_blank">S2 Table</a>.</p