Ramon Jordan

Comparison and differentiation of potyvirus isolates and identification of strain-, virus-, subgroup-specific and potyvirus group-common epitopes using monoclonal antibodies

A panel of monoclonal antibodies (MAbs) generated against an admixture of 12 potyvirus isolates was used to compare and differentiate diverse potyviruses. Both native and denatured virions of strains of bean yellow mosaic (BYMV), potato virus Y, tobacco etch, pea seed-borne mosaic, iris severe mosaic, iris mild mosaic and asparagus virus-1 potyviruses were used as immunogen and as antigen for screening of the hybridoma cell lines. Thirty cell lines secreting potyvirus-specific antibodies reactive in indirect antigen-coated plate (ACP-) ELISA were selected for detailed analysis. All 30 MAbs reacted with at least one strain of BYMV; 11 MAbs reacted with between one and eight of the nine BYMV strains and an additional three MAbs reacted only with isolates within the BYMV subgroup (BYMV, pea mosaic virus and clover yellow vein virus). The remaining 16 MAbs reacted with a BYMV isolate and with at least one of the other 43 potyvirus isolates tested. MAb PTY 1 reacted with all 55 potyvirus isolates tested (representing at least 33 different and distinct aphid-transmissible potyviruses). The potyvirus cross-reactive MAbs generally gave higher reactivity values in ACP-ELISA with dissociated virus than with polyclonal antibody-trapped intact virions in triple antibody sandwich ELISA (i.e. were cryptotope-specific). The BYMV strain- and virus-specific MAbs reacted strongly with both types of antigens (i.e. were metatope-specific). At least 25 distinct epitopes (12 cryptotopes and 13 metatopes) could be identified from the MAb-antigen reactivity patterns. The distribution of these epitopes between virus isolates can be used to detect and differentiate potyviruses in infected plant extracts and to examine virus architectures. Some of these epitopes are shared by potyvirus isolates not previously shown to be serologically related. The broad spectrum-reacting MAb PTY 1 recognizes a cryptotope conserved on all of the aphid-transmissible potyviruses examined and should be a valuable tool for the detection and assay of these potyviruses.

Two unique US isolates of Pepino mosaic virus from a limited source of pooled tomato tissue are distinct from a third (European-like) US isolate

Summary.Three strains of Pepino mosaic virus (PepMV) found in the US have been cloned and sequenced by RT-PCR using total RNA from infected tissue as template, and degenerate potexvirus- and PepMV species- and isolate-specific primers. Despite limited source material, the complete nucleotide sequences (6413 and 6410 nts, respectively) of two isolates, PepMV-US1 and PepMV-US2, were obtained and analyzed using total RNA from less than 0.2 g of a pooled infected tomato leaf sample from Arizona. Sequence of the 3′-end of the third isolate from infected fresh tomato fruits from Maryland (PepMV-US3) was also determined. The genome organizations of PepMV-US1 and US2 were typical of the genus Potexvirus, with the following reading frame order: ORF 1, encoding a putative replicase; ORFs 2–4, triple gene block proteins (TGBp) 1–3; and ORF 5, coat protein (CP). Gene-for-gene comparison between PepMV-US1 and US2 revealed the following amino acid identities: 91% in replicase, 89% in TGBp1, 92% in TGBp2, 85% in TGBp3, and 93% in the CP; with an overall nucleotide identity of 86%. Nucleotide sequence comparisons between US1 and US2 and the European isolates showed only 79–82% identity, whereas the identity among the European isolates was over 99%. Sequence comparisons and phylogenetic analysis indicate that PepMV-US1 and US2 are distinctly different from the European isolates, while the CP of PepMV-US3 is nearly identical to the European isolates. The results presented also suggest that TGBp1 and TGBp3 are more suitable than either the replicase or coat protein gene products for discriminating PepMV isolates.

Transgenic Approaches to Disease Resistance in Ornamental Crops

Abstract Viral, bacterial, and fungal diseases of ornamental plants cause major losses in productivity and quality. Chemical methods are available for control of fungal diseases, and to a lesser extent for bacterial diseases, but there are no economically effective chemical controls for viral diseases except to control vector species. Host plant resistance is an effective means of controlling plant diseases, and minimizing the necessity for the application of pesticides; however, there are many ornamentals in which no natural disease resistance is available. It is possible to introduce resistance derived from other species, or even from the pathogen itself, by genetic engineering. This allows the introduction of specific, or in some instances broad spectrum, disease resistance into plant genotypes that have been selected for desirable horticultural characters; in contrast, introduction of natural resistance by traditional breeding may take many cycles of breeding to combine disease resistance with desirable ornamental quality. This article briefly reviews existing work on transformation systems for ornamentals, and discusses the various approaches to introducing resistance to viral, bacterial, and fungal diseases, and to nematode infestations. These include pathogen-related proteins, R genes, and general pathogen resistance; anti-microbial peptides; expression of anti-pathogen antibodies; viral sequences; ribozymes; antiviral peptides; ribonucleases; and ribosome-inactivating proteins. Examples are given of application of these approaches to disease resistance in other types of crop and model plant systems, and actual or potential application to disease resistance in ornamentals. Future prospects for obtaining plants with multiple pest and disease resistances are discussed.

Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display

Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierces disease of grapevine.

The complete nucleotide sequence, genome organization, and specific detection of Beet mosaic virus

Summary.Beet mosaic virus (BtMV) was identified almost five decades ago but has not been fully characterized at the molecular level. In this study, we have determined for the first time the complete nucleotide sequence of BtMV genomic RNA and have developed a specific molecular means for its diagnosis. The viral genome of BtMV comprises 9591 nucleotides, excluding the 3′ terminal poly (A) sequence, and contains a single open reading frame (ORF) that begins at nt 166 and terminates at nt 9423, encoding a single polyprotein of 3086 amino acid residues. A 3′ untranslated region of 168 nucleotides follows the ORF. The deduced genome organization is typical for a member of the family Potyviridae and includes 10 proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb and coat protein (CP). Nine putative protease cleavage sites were predicted computationally and by analogy with genome arrangements of other potyviruses. Conserved sequence motifs of homologous proteins of other potyviruses were found in corresponding positions of BtMV. BtMV is a distinct species of the genus Potyvirus with the most closely related species being Peanut mottle virus (∼55% amino acid identity). Based on the nucleotide sequence obtained, we have developed a virus-specific RT-PCR assay for accurate diagnosis and differentiation of BtMV.

Complete nucleotide sequence of clematis chlorotic mottle virus, a new member of the family Tombusviridae

Clematis chlorotic mottle virus (ClCMV) is a previously undescribed virus associated with symptoms of yellow mottling and veining, chlorotic ring spots, line pattern mosaics, and flower distortion and discoloration on ornamental Clematis. The ClCMV genome is 3,880 nt in length with five open reading frames (ORFs) encoding a 27-kDa protein (ORF 1), an 87-kDa replicase protein (ORF 2), two centrally located movement proteins (ORF 3 and 4), and a 37-kDa capsid protein (ORF 5). Based on morphological, genomic, and phylogenetic analysis, ClCMV is predicted to be a member of the genus Pelarspovirus in the family Tombusviridae.

Detection of Jasmine virus H and characterization of a second pelarspovirus infecting star jasmine (Jasminum multiflorum) and angelwing jasmine (J. nitidum) plants displaying virus-like symptoms

Star jasmine (Jasminum multiflorum) plants growing in Hawaii expressing a diverse array of virus-like foliar symptoms were examined for the presence of a causal agent. Symptomatic tissues collected from three locations on the island of Oahu, Hawaii consistently harbored double-stranded (ds)RNAs approximately 4.2 and 1.7 kbp in size. Sanger and high-throughput sequencing approaches revealed these dsRNAs were from two distinct virus species co-infecting the same host plant. One of these two viruses was the recently characterized Jasmine virus H (JaVH), and the second we designated as Jasmine mosaic-associated virus (JMaV). Both viruses were subsequently found, by high-throughput sequencing, in a single angelwing jasmine (J. nitidum) plant exhibiting similar ringspot symptoms and growing at the U.S. National Arboretum in Washington, DC. Phylogenetic placement, genome organization, and sequence comparisons indicate these two viruses are classifiable as members of the genus Pelarspovirus (family Tombusviridae). To determine if either of these viruses were associated with the observed symptoms, a PCR-based detection assay was developed to detect and distinguish these two viruses in several Hawaii-grown plants. All 32 samples collected from four Oahu locations displayed symptoms. All 32 samples were positive for JaVH, and 16 were positive for JMaV. An asymptomatic star jasmine plant from the island of Hawaii was negative for both JaVH and JMaV. Both viruses were also found in a symptomatic J. sambac sample from Maryland while only JMaV was detected in a symptomatic Jasminum sp. sample from California.