An efficient papaya leaf distortion mosaic potyvirus vector for virus-induced gene silencing in papaya

Papaya (Carica papaya L.) is regarded as an excellent model for genomic studies of tropical trees because of its short generation time and its small genome that has been sequenced. However, functional genomic studies in papaya depend on laborious genetic transformations because no rapid tools exist for this species. Here, we developed a highly efficient virus-induced gene silencing (VIGS) vector for use in papaya by modifying an artificially attenuated infectious clone of papaya leaf distortion mosaic virus (PLDMV; genus: Potyvirus), PLDMV-E, into a stable Nimble Cloning (NC)-based PLDMV vector, pPLDMV-NC, in Escherichia coli. The target fragments for gene silencing can easily be cloned into pPLDMV-NC without multiple digestion and ligation steps. Using this PLDMV VIGS system, we silenced and characterized five endogenous genes in papaya, including two common VIGS marker genes, namely, phytoene desaturase, Mg-chelatase H subunit, putative GIBBERELLIN (GA)-INSENSITIVE DWARF1A and 1B encoding GA receptors; and the cytochrome P450 gene CYP83B1, which encodes a key enzyme involved in benzylglucosinolate biosynthesis. The results demonstrate that our newly developed PLDMV VIGS vector is a rapid and convenient tool for functional genomic studies in papaya.

Nanopore sequencing of tomato mottle leaf distortion virus, a new bipartite begomovirus infecting tomato in Brazil

During a survey in a tomato field in Luziânia (Goiás State, Brazil), a plant showing mottling, chlorotic spots, and leaf distortion was found. A new bipartite begomovirus was found by Nanopore sequencing, and the full DNA-A sequence was confirmed by Sanger sequencing. The highest nucleotide identity match of this DNA-A genome (2596 bases) was 81.65% with tomato golden leaf deformation virus (HM357456). Due to the current species demarcation criterion of 91% of nucleotide identity (DNA-A), we propose it as a new member of the genus Begomovirus, named Tomato mottle leaf distortion virus.

Partial sequencing of a putative Alstroemeria necrotic streak orthotospovirus isolate detected on lettuce in Colombia

ABSTRACT Lettuce is the most cultivated leafy salad vegetable in Colombia, being the municipality of Madrid, in the Department of Cundinamarca, the second largest producer. In this region, lettuce plants with foliar symptoms characterized by brown necrotic spots forming an extended necrotic area, chlorosis, leaf distortion and plant stunting have been detected, possibly caused by a viral infection associated with the Orthotospovirus genus. This study aimed to identify the orthotospovirus species associated with those symptoms, contributing to updating the lettuce phytosanitary status in this region. The presence of orthotospovirus was confirmed by the enzyme-linked immunosorbent assay (DAS-ELISA), although the sequence of the nucleocapsid (N) gene confirmed the presence of Alstroemeria necrotic streak orthotospovirus, disregarding the Tomato spotted wilt orthotospovirus and Impatiens necrotic spot orthotospovirus previously reported for this crop, being this its first report in lettuce crops in Colombia.

Interactive effects of Potato virus Y and Potato leafroll virus infection on potato yields in Uganda

Potatoes are prone to attack by multiple viruses, which contribute greatly to yield and quality decline depending on the cultivar and the virus involved. This study investigated the effect of co-infection involving Potato virus Y (potyvirus) and Potato leafroll Virus (pelero virus) on productivity of five potato cultivars in Uganda and the nature of virus interaction during co-infection process. Variety response to virus infection by PVY, PLRV and co-infection (PVY + PLRV) varied across different varieties. The plants that were infected with PLRV had leaf rolling, stuntedness, leaf distortion, reduction in leaf size and mottling and light yellow mosaics, and in some cases, purple or red margins were observed, while single infection of PVY induced necrosis, leaf rugosity, crinkling, stunting, interveinal necrosis, blotching of the margins, leaf distortion and mottling. When the two viruses were combined during co-infection with PVY + PLRV, the symptoms were characterized by bright blotching and necrotic leaf margins with purpling of the leaf tips and leaf margins, stuntedness and leaf distortions. The virus disease severity was higher under mixed infected plants than single infected plants. The high disease severity culminated in a significant effect on yield, marketable tuber number per plant, plant growth height and plant vigor, which were different across the varieties. Co-infection involving PVY and PLRV caused a reduction in the marketable yield of 95.2% (Kinigi), 94% (Victoria), 89.5 (Rwagume), 45.3% (Royal) and 23.7% (Sifra). Single infection by PLRV caused a reduction in a marketable yield in Victoria (91.8%), Kinigi (84.8%), Rwagume (73.3%), Royal (47.2%) and Sifra 22.1%, while PVY caused a marketable yield reduction in Victoria (87.2%), Rwagume (85.9.7%), Kinigi (85.1%), Royal (37.4%) and Sifra (14.1%). The effects associated with the co-infection of PVY and PLRV were lower than the combined value of the single infections, suggesting that the two viruses were interacting to affect the potato productivity. The high yield loss suggested that effective resistance strategy targeting PVY, PLRV and their combination was required to save the potato industry in Uganda.

A Double Mutation in the Conserved Motifs of the Helper Component Protease of Papaya Leaf Distortion Mosaic Virus for the Generation of a Cross-Protective Attenuated Strain

Potyviral helper component protease (HC-Pro), as a major determinant of symptom expression in susceptible plants, is a likely target candidate in the production of attenuated strains for cross-protection. In this study, single or double mutations of Lys (K) to Glu (E) in the Lys-Ile-Thr-Cys motif and Arg (R) to Ile (I) in the Phe-Arg-Asn-Lys motif of the HC-Pro from the severe papaya leaf distortion mosaic virus strain DF (PLDMV-DF) reduced symptom expression and virus accumulation in infected papaya (Carica papaya) plants. The papaya plants infected with the attenuated double mutant of PLDMV-EI presented as symptomless. PLDMV-EI provided effective protection against PLDMV-DF infection in three papaya cultivars and had no effect on plant growth and development. Our result showed that PLDMV-EI is a promising mild strain for the practical use of cross-protection in the field.

Partial molecular characterization of begomoviruses isolated from weeds collected in tomato crops in the southeast of Valle del Cauca, Colombia

Weeds are usually considered as a source of new viruses but are often neglected during diversity studies. Previously eleven samples of weeds were collected along the edges of a tomato crop field located in four municipalities (Florida, Ginebra, Cerrito and Candelaria) in the Southeast of Valle del Cauca. These samples were positive for begomoviruses but their molecular characterization had not been done until now. For each sample, DNA fragments were cloned, sequenced and analyzed. Nucleotide sequence analysis of viral fragments showed the presence of six different begomoviruses: two virus isolated from Lantana camara, Desmodium sp. and Amaranthus dubius were previously described as potato yellow mosaic virus (PYMV) and passionfruit leaf distortion virus (PLDV), respectively; other four virus which were isolated from L. camara, A. dubius, Rivina humilis, Desmodium sp., Rhynchosia minima, Hybanthus attenuatus, Verbena sp., Croton hirtus and Caesalpinia sp., showed its highest nucleotide sequence identity (89%) with bean chlorotic mosaic virus (BChMV), datura leaf distortion virus (DaLDV) and rhynchosia golden mosaic Yucatan virus (RhGMYV). Cloned viruses fragments from these weeds could be new begomoviruses not previously reported before, this in accordance with the ICTV species demarcation criterion for the genus Begomovirus (≥91 % sequence identity). Mixed infections of begomoviruses in Desmodium sp. and A. dubius weeds also was found in this analysis. Finally, this paper reports by the first time to three alternative begomoviruses weeds hosts that infect tomato and passion fruit crops: A. dubius for PYMV, and L. camara and Desmodium sp. for PLDV, respectively.

Thladiantha Dubia Mosaic Virus: A Novel Potyvirus Infecting Manchurian Tubergourd (Thladiantha dubia) in Northeast China

A new virus with flexuous, filamentous particles approximately 650 nm long was discovered in Manchurian tubergourd (Thladiantha dubia Bunge) leaves exhibiting severe mosaic symptoms. The whole genome sequence of the virus was determined by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The entire genome consisted of 10,112 nucleotides (nt) excluding the poly (A) tail, which shared the highest nucleotide sequence identity (73.8%) with that of papaya leaf distortion mosaic virus Hainan-DF isolate (PLDMV-Hainan-DF). A phylogenetic analysis showed that this virus clustered with PLDMV isolates in a subbranch within the potyviral clade. Of the 23 species of indicator plants tested, only potato and its original host were systemically infected by the virus tested upon mechanical inoculation. A field survey showed that the virus was widely distributed on T. dubia and potatoes in Northeast China. Moreover, this virus displayed a high degree of genetic variation as evaluated by the sequences of the coat protein (CP) gene. Based on these results, the name Thladiantha dubia mosaic virus (ThDMV) is proposed for this new potyvirus.