A transient expression system using onion epidermal cells was used to investigate domains of the Tobacco mosaic virus (TMV) 126-kDa replicase protein involved in cellular localization. Initially, a nuclear localization signal (NLS), identified within the amino-terminus of the 126-kDa protein, was investigated for its functionality using fusion constructs containing the green fluorescent protein (GFP). Fusion of the amino-terminal 70 amino acids of the 126-kDa protein, containing the NLS, to a beta-glucuronidase-GFP open reading frame (ORF), directed the accumulation of fluorescence to the nucleus. In contrast, similar constructs lacking the NLS or containing a mutated NLS sequence failed to accumulate within the nucleus. Additional investigations using GFP fusion constructs containing the first 178 or 388 amino acids of the 126-kDa protein also displayed nuclear localization. However, fusion constructs encoding the first 781 amino acids or the entire 126-kDa ORF did not accumulate within the nucleus but instead associated with the endoplasmic reticulum (ER), forming spot-like inclusions. Thus, a dominant ER association domain exists between amino acids 388 and 781 of the 126-kDa protein. Interestingly, a full-length 126-kDa GFP fusion construct encoding a nonfunctional NLS mutation also localized to the ER but did not form inclusions. Furthermore, a TMV mutant containing the same nonfunctional NLS mutation failed to replicate in protoplasts. Together these findings suggest that both the NLS and the ER retention domain contribute to the functional localization of the 126-kDa protein.

Resumo -O objetivo deste trabalho foi determinar a presença do alelo Pvr4, que confere resistência contra o PepYMV (Pepper yellow mosaic virus), em genótipos de pimentão comunmente encontrados no mercado brasileiro, com uso de um marcador molecular codominante tipo CAPS. A resistência ao PepYMV, nos genótipos CM-334-INRA, Myr-29 e em genótipos derivados do híbrido comercial Mônica-R, foi detectada como associada à banda de 444 pb, ligada ao alelo de resistência Pvr4. As plantas resistentes homozigotas (Pvr4/Pvr4) mostraram uma banda de 444 pb, as suscetíveis (Pvr4 Marker-assisted selection for resistance to potyvirus in sweet pepperAbstract -The objective of this work was to determine the presence of the Pvr4 allele, which controls the resistance to the PepYMV (Pepper yellow mosaic virus), in sweet pepper genotypes commonly available in the Brazilian market, using a CAPS codominant molecular marker. The resistance to PepYMV, in the genotypes CM-334-INRA, Myr-29, and in genotypes derived from the hybrid Mônica-R, was found to be associated with the 444 bp band linked to the resistance allele Pvr4. Homozygous resistant plants (Pvr4/Pvr4) showed a single band of 444 bp, the susceptible ones (Pvr4showed a band of 458 bp, and the heterozygous resistant plants (Pvr4 + /Pvr4) showed both bands. However, in the resistant accession CM-334-UFV, and in the hybrids Magali-R and Martha-R, as well as in populations derived from this accession and these hybrids, the resistance to PepYMV was not associated to the CAPS marker. The accession CM-334-UFV ('Criollo de Morelos-334' from Viçosa, MG, Brazil) was distinct from CM-334-INRA ('Criollo de Morelos-334', from France); although both accessions were resistant to PepYMV, the association of resistance with the 444 bp band was found only in CM-334-INRA.

-specific monoclonal antibodies did not recognize PVY-AGA. The ability of these new PVY recombinants to overcome resistance genes in potato producing mild or no symptoms, combined with the lack of serological reactivity towards at least one PVY N -specific antibody may present a significant threat posed by these isolates to seed potato production areas.

In Brazil, Potato virus Y (PVY) currently presents a significant problem for potato production, reducing tuber yield and quality. Recombinant tuber necrotic isolates of PVY had been reported to occur in the country but no systematic study of the PVY isolate diversity was conducted thus far. Here, a panel of 36 PVY isolates, randomly collected in Brazil from potato between 1985 and 2009, was subjected to a systematic molecular and serological typing using reverse-transcription polymerase chain reaction and a series of PVYO- and PVYN-specific monoclonal antibodies. The data collected were combined with biological characterization of the same isolates in tobacco. Of the 36 isolates tested, 3 were typed as PVYO, 10 as PVYN:O/N-Wi, 21 as PVYNTN, and 2 as “unusual” or inconclusive. Of the 10 isolates from the recombinant PVYN:O/N-Wi strain group, 1 isolate, MAF-VOY, was found to have an unusual serological profile identical to the nonrecombinant PVYO-O5 strain group. The 21 tested PVYNTN isolates included 1 isolate that did not induce vein necrosis in tobacco and 2 isolates with an unusual serological profile (i.e., displaying negative reactivity to one commercial PVYN-specific monoclonal antibody). Whole genome sequences were determined for four PVY isolates from Brazil, representing PVYO, PVYNTN, and PVYN-Wi strains. The genome of the MAF-VOY isolate was found to be recombinant, having characteristic N-Wi structure with two recombinant junctions and carrying a single mutation in the capsid protein at position 98, which led to an unusual O5 serological reactivity. Taken together, the data obtained suggest that the two recombinant strains, PVYNTN and PVYN:O/N-Wi, now are apparently dominant in the Brazilian potato crop. The data also suggest that recombinant isolates in Brazil often have unusual serological reactivity which may hamper their correct identification by conventional typing based on enzyme-linked immunosorbent assay.

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