Detection of passion fruit green spot virus in plant hosts and evaluation of the 5' end of its RNA2

Abstract

Brazil is the largest producer and consumer of passion fruit (Passifora spp.) in the world, including the use of products derived from the fruit. The crop generates thousands of jobs, directly and indirectly, in addition to being an important source of income for small and medium producers. The passion fruit is affected by several diseases, including "definhamento precoce" and green spot of passion fruit, first reported in Brazil in the 1990s.Both are caused by passion fruit green spot virus (PfGSV) and are characterized by the appearance of chlorotic spots on the leaves, green spots on the fruits and intense necrosis on the branches. The pathogen is transmitted by the mite Brevipalpus yothersi and its management is carried out exclusively by the chemical control of its vector.PfGSV, belonging to the genus Cilevirus, family Kitaviridae, has a bisegmented single-stranded RNA genome of positive sense with ~5 and ~9 kb for RNA1 and RNA2, respectively. Like all cileviruses, PfGSV replicates and causes characteristic cytopathic effects in the cytoplasm of infected cells, and are considered Brevipalpus mites transmitted viruses (BTV) of the cytoplasmic-type or BTV-C. The recent characterization of five PfGSV isolates not only confirmed their taxonomic classification as a cilevirus but also evidenced the presence of two ORFs (open reading frames) that do not have nucleotide sequence identity with other ORFs present in international databases such as GenBank. These ORFs, called p11 and p13, were recognized as orphan ORFs (ORFan). The p11 and p13 ORFans are close to the 5' end of RNA2, at a locus known as the intergenic region (IR).Due to the presence of ORFans and small ORFs that encode proteins with transmembrane domains, the IR of cileviruses shows a marked polymorphism and corresponds to an interesting region to be studied. The PfGSV genome has seven ORFs, being RdRp (RNA-dependent RNA polymerase) and p29 (putative coat protein) in RNA1 and p15 (unknown function), p13/p11 (unknown function), p61 (possible glycoprotein), MP (movement protein) and p24 (unknown function) in RNA2.This project is a continuation of the project developed by the student Isabela Leão during her CNPq/Pibic scholarship (from August 2020 - August 2021). So far, PfGSV has been detected in approximately 20 samples from different Brazilian localities by RT-PCR using three specific primer pairs. Of these, orchid plants were sequenced by high-throughput sequencing (HTS) using the Illumina platform (San Diego, USA). The general objective of this proposal is to characterize the 5' end of PfGSV RNA2 not only from passion fruit samples, but also from other plant hosts, including orchids. (AU)