Xianzhou Nie

A cereal haemoglobin gene is expressed in seed and root tissues under anaerobic conditions

Legumes, and a very few non-legume plant species, are known to possess functioning haemoglobin genes. We describe here the characterization of a haemoglobin cDNA isolated from barley. The deduced amino acid sequence shows 71% amino acid identity with a non-legume haemoglobin gene, a further 16% of the residues being conservative replacements. The barley cDNA also hybridizes to genomic sequences in rye, maize and wheat. The demonstration of a gene from a monocotyledon with close sequence homology to the known non-legume plant haemoglobins fills a major gap in the known distribution of haemoglobin genes in the plant kingdom. The expression of the gene is induced in isolated barley aleurone layers exposed to anaerobic conditions, and the roots of flooding-stressed barley plants. The expression of the RNA under anoxic conditions is similar to that of a known anaerobic response gene, alcohol dehydrogenase. Our results suggest that the increased expression of haemoglobin RNA is an integral part of the normal anaerobic response in barley. The findings are discussed in the light of current theories of haemoglobin function and evolution.

Diversity Among Potato virus Y Isolates Obtained from Potatoes Grown in the United States

ABSTRACT Potato field isolates (Solanum tuberosum) of Potato virus Y (PVY) collected from the midwestern and western United States were characterized using serological, molecular, and biological assays. PVY field isolates were grouped into the previously defined categories: PVY(O), European PVY(NTN), North American PVY(NTN), and PVY(N:O) recombinant and four previously undefined groups. Studies reported here agree with published reports from Europe and elsewhere in North America as PVY isolates capable of causing veinal necrosis in tobacco indicator plants appear in high frequency. In contrast to European experiences, PVY tuber necrosis isolates have a PVY(O) coat protein rather than that of PVY(N). Several PVY(N:O) recombinant isolates induced potato tuber necrotic ringspot disease (PTNRD) in the highly susceptible potato cv. Yukon Gold. The PTNRD symptoms produced by these PVY(N:O) recombinants were atypical compared with lesions found on the same cultivar infected with either the European or North American PVY(NTN) isolates. These PVY(N:O) isolates produced a roughly circular, sunken necrotic lesion on the surface of the tuber instead of the typical external sunken ring pattern displayed by PVY(NTN) isolates. This study establishes the complex nature of PVY populations within the U.S. potato industry and clearly demonstrates the diverse nature of PVY in the United States.

A new approach for the simultaneous differentiation of biological and geographical strains of Potato virus Y by uniplex and multiplex RT-PCR.

Two strains of Potato virus Y (PVY), the common (PVY(O)) and the tobacco veinal necrosis (PVY(N)) have been known for decades. More recently, a tuber ringspot necrosis (PVY(NTN)), and several recombinants of PVY(O) and PVY(N) (designated here as PVY(N:O)) have been described. Further, the PVY(N) group of strains have been assigned to two geographical subgroups of European (EU) PVY(N/NTN) and the North American (NA) PVY(N/NTN). The evolution of new PVY(N) strains, has complicated the diagnosis, which requires a combination of bioassay, serological and molecular assays. To simplify the identification and differentiation of various PVY(N) strain groups, a competitive (single antisense and multiple sense primers) reverse transcription-polymerase chain reaction (RT-PCR) was used, making use of minor differences in the variable region part of the PVY genome. Specifically, primers based on small variations in nucleotide stretches of P1 gene permitted a broad range separation of PVY(O) and PVY(N) groups and the specific detection of strain subgroups. The primer pairs designed for identifying PVY(O), EU-PVY(N/NTN), NA-PVY(N) and NA-PVY(NTN) are described. Primer pairs can be used in a uniplex (single pair of primer) or multiplex (duplex, tetraplex or pentaplex) competitive RT-PCR, allowing simultaneous testing for any combination of PVY(O), EU-PVY(N/NTN), NA-PVY(N) and NA-PVY(NTN).

Mitochondrial Respiration and Hemoglobin Gene Expression in Barley Aleurone Tissue

Previous studies have shown that plant hemoglobin (Hb) mRNA is expressed in barley (Hordeum vulgare L.) aleurone layers during hypoxia. We have examined the effect of a number of respiratory inhibitors on barley aleurone layers to determine the factors that influence Hb gene expression. Respiratory inhibitors that reduce O2 consumption, such as CO, cyanide, and antimycin A, strongly enhanced Hb mRNA levels. Treatment with the oxidative phosphorylation uncoupler 2,4-dinitrophenol markedly increased O2 consumption and had a similar positive effect on Hb gene expression. Hb transcript levels were also stimulated by the ATP synthase inhibitor oligomycin. The results suggest that the expression of Hb is not directly influenced by O2 usage or availability but is influenced by the availability of ATP in the tissue.

Specific differentiation of recombinant PVYN:O and PVYNTN isolates by multiplex RT-PCR

The recombinant isolates of tobacco veinal necrotic strain of Potato virus Y (PVYN) and potato tuber necrotic group (PVY(NTN)) contain segments of the PVYO and the PVY(N) genome. Three major recombinant junctions (RJ) are present in the genome of the recombinant PVY(NTN) at sites HC/Pro-P3, 6K2-NIa, and the C-terminal region of CP gene and one RJ at HC/Pro-P3 site in some recombinant PVYN isolates (termed PVY(N:O)). Protocols for specific differentiation of the recombinant PVY(NTN) and PVY(N:O) from the non-recombinant PVYN are described. Specific primer pairs were designed to target the three RJs so that sense and antisense primers completely matched the nucleotide sequences at either side of the RJ. In a uniplex reverse transcription-polymerase chain reaction (RT-PCR), the first primer pair amplified a fragment of 641bp from the recombinant PVY(NTN) and PVY(N:O). The second and third primer pairs exclusively amplified fragments of 448 and 290bp, respectively from the recombinant PVY(NTN). In a multiplex (triplex) RT-PCR, when all three primer pairs were used simultaneously, the three fragments (641, 448 and 290bp) were amplified exclusively from the recombinant PVY(NTN), while only one fragment (641bp) was amplified from the PVY(N:O) isolates, clearly differentiating the two recombinant isolates. No amplification was observed from the non-recombinant PVY, including PVYO and North American (NA)-PVY(N/NTN). For further improvement of the multiplex RT-PCR, effects of cDNA preparation using specific antisense primers, random primers or oligo(dT) plus random primers were investigated. The cDNA prepared by random primer plus oligo(dT) increased the overall band intensity.

Reverse Transcription Loop-Mediated Isothermal Amplification of DNA for Detection of Potato virus Y

A reverse transcription loop-mediated isothermal amplification of DNA (RT-LAMP) for detection of Potato virus Y (PVY) was developed. In this procedure, a set of four primers matching a total of six sequences of the coat protein (CP) gene of PVY were designed in such a way that a loop could be formed and elongated during DNA amplification. Using PVY CP complementary DNA clones as templates, the LAMP reaction was optimized by adjusting the concentrations of MgSO4, dNTPs, and Bst DNA polymerase. The effects of fragment length of target DNA on LAMP also were investigated. Two-step and one-step RT-LAMPs were performed using RNA extracts of various PVY cultures, and the results were correlated with two-step reverse transcription polymerase chain reaction (RT-PCR) for detection of PVY. Further, the turbidity caused by precipitation of magnesium pyrophosphate formed in positive RT-LAMP reactions was used to measure the amplification by utilizing a time-saving spectrophotometric method. The one-step RT-LAMP-turbidity method gave results comparable with the two-step RT-PCR method for detection of PVY from potato leaf and tuber samples. Of the total 240 samples, 234 were diagnosed similarly by both methods.

Possible Escape of a Recombinant Isolate of Potato virus Y by Serological Indexing and Methods of its Detection

Surveys of commercial and seed potato fields for virus diseases (1998 to 2002) in Manitoba established that Potato virus Y (PVY) is of concern in seed potato production. To determine the prevalence of PVY strains, PVY-infected tubers identified by reverse transcription-polymerase chain reaction (RT-PCR) from surveys (2000 to 2001) were grown for symptom expression and strain characterization by strain-specific RT-PCR, bioassays, and serological assays. Of the samples collected (2000 to 2001) and tested by RT-PCR, 4.0% contained PVY. Further analysis of the PVY-positive samples by a duplex RT-PCR facilitating the simultaneous detection of common (PVYO) and tobacco veinal necrosis strains (PVYN/NTN) indicated that 37.5% contained PVYO and 63.5% contained PVYN-type isolates. Analysis of the PVYN-type samples using three monoclonal antibodies (MAbs) showed that all reacted with only the PVYO MAbs and not with the PVYN-specific MAb. Partial nucleotide sequences of both ends of PVY-RNA showed that the PVYN-type isolates resembled those reported in 1996 from Manitoba. These isolates are designated as PVYN:O. In view of the increased incidence of PVYN:O in one production area, seed tubers imported from other provinces of Canada and the neighboring United States were analyzed for PVYN:O. The PVYN:O was detected in imported seeds from Minnesota, Montana, and North Dakota.

Detection of multiple potato viruses using an oligo(dT) as a common cDNA primer in multiplex RT-PCR

A novel usage of multiplex reverse transcription polymerase chain reaction (m-RT-PCR) for simultaneous detection of multiple viruses is reported. By use of an oligo(dT), as a common primer, nearly full-length cDNAs can be synthesized. Furthermore, combining an oligo(dT) primer with a specific antisense primer can be used to simultaneously prime reverse transcription of both polyadenylated and non-polyadenylated RNAs. Four viral genera including five potato viruses [(carlavirus (PVS), polerovirus (PLRV), potexvirus (PVX), potyvirus (PVA and PVY))] and a viroid genus including a viroid genome (pospiviroid (PSTVd)) were used to develop various formats of m-RT-PCR. In artificially created viral RNA mixtures, all six RNA pathogens were detected successfully by uniplex- and m-RT-PCR. In naturally infected field grown tubers, m-RT-PCR detected infection of two to three viruses, which were present in the tubers.

Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR.

Phenolic compounds from plant tissues inhibit reverse transcription-polymerase chain reaction (RT-PCR). Multiple-step protocols using several additives to inhibit polyphenolic compounds during nucleic acid extraction are common, but time consuming and laborious. The current research highlights that the inclusion of 0.65 to 0.70% of sodium sulphite in the extraction buffer minimizes the pigmentation of nucleic acid extracts and improves the RT-PCR detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) in potato (Solanum tuberosum) tubers and Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) in leaves and bark in the sweet cherry (Prunus avium) tree. Substituting sodium sulphite in the nucleic acid extraction buffer eliminated the use of proteinase K during extraction. Reagents phosphate buffered saline (PBS)-Tween 20 and polyvinylpyrrolidone (PVP) were also no longer required during RT or PCR phase. The resultant nucleic acid extracts were suitable for both duplex and multiplex RT-PCR. This simple and less expensive nucleic acid extraction protocol has proved very effective for potato cv. Russet Norkotah, which contains a high amount of polyphenolics. Comparing commercially available RNA extraction kits (Catrimox and RNeasy), the sodium sulphite based extraction protocol yielded two to three times higher amounts of RNA, while maintaining comparable virus detection by RT-PCR. The sodium sulphite based extraction protocol was equally effective in potato tubers, and in leaves and bark from the cherry tree.

Evolution of North American PVYNTN Strain Tu 660 from Local PVYN by Mutation rather than Recombination

A North American (NA) isolate of tobacco veinal necrotic strain of Potato virus Y (PVYN) (N-Jg) and a NA isolate of potato tuber necrotic strain of Potato virus Y (PVYNTN) (Tu 660) were tested for their phenotypes by inoculation to potato plants of three potato cultivars. Upon inoculation with Tu 660, tubers of the cultivars ‘Norchip’ and ‘Ranger Russet’ developed potato tuber necrotic ringspot disease (PTNRD) but not the tubers of ‘Russet Burbank’. N-Jg failed to induce PTNRD in the tested cultivars. The genomic RNAs of both strains were completely sequenced and analysed. High homology (98% and 99% identity on nucleotide and polyprotein, respectively) was found between Tu 660 and N-Jg. When polyproteins were compared with other isolates, high identity was observed between Tu 660 and an European (Eu) PVYN-605 (98%) and with an Eu-PVYNTN-H (96%). However, when individual mature proteins were compared, much lower identities (86.5–94%) were found between Tu 660 and PVYNTN-H compared to 98–99.5% between Tu 660 and PVYN-605 in the P3, 6K1 and CI regions. Further sequence analysis indicated that the PVYNTN-H is a hybrid molecule of the genomic RNA recombination of PVYO and Eu-PVYN as shown by Glais et al. (Arch Virol 147, 363–378), whereas NA-PVYNTN Tu 660 is free of recombination points. Phylogenetic analysis confirmed this observation, and suggested that, in light of high homology, the Tu 660 might have evolved from NA-PVYN by mutations rather than the genome recombinations. The non-recombinant nature of NA-PVYNTN Tu 660 strongly suggests that the recombinant structure of genome is not a necessary prerequisite for the PTNRD phenotype.