No description available.

Complementary DNA (cDNA) clones representing approximately 5800 nucleotides of potato leafroll virus (PLRV) genomic RNA were generated, restriction-mapped, and partially sequenced. Within one of the cDNA clones an open reading frame (ORF) that could encode a 23 kDa protein was identified and further characterized. Comparison of the deduced amino acid sequence with the coat protein amino acid sequence of the PAV strain of barley yellow dwarf luteovirus (BYDV-PAV) showed significant similarity. This observation together with its size and internal location within the genome suggested that this gene encoded the PLRV coat protein. Other similarities were observed between PLRV and BYDV sequences in this region of their genomes, including a 17 kDa ORF within the ORF encoding the 23 kDa coat protein, and termination of the latter with an amber codon which is immediately followed by a large ORF in the same reading frame. Three PLRV coat protein gene sequences were used to transform tobacco and the potato cultivars 'Desiree' and 'Russet Burbank' via Agrobacterium tumefaciens mediated gene transfers. One construct possessed 12 nucleotides of the untranslated leader sequence 5' to the putative coat protein gene start codon. The other construct, which was also inserted in the reverse orientation to produce negative-sense RNA, had 192 nucleotides from this leader sequence. When these sequences were introduced as chimaeric genes under the control of a duplicated cauliflower mosaic virus 35S (CaMV) promoter, transcription levels were high. Both positive-sense transcripts produced potato leafroll coat protein which accumulated to maximum levels of approximately 0.5% and 0.01% of total leaf protein in tobacco and potato, respectively. Results show that significant levels of inoculum concentration-independent sustained resistance were obtained with each construct, resulting in PLRV titres as low as 1% of the level observed in untransformed plants, as determined by enzyme-linked immunosorbent assays. Virus transmission from PLRV-inoculated transgenic 'Russet Burbank' was reduced substantially and was correlated with virus titre. The pattern and level of protection were the same for constructs producing positive- and negative-sense RNA, suggesting a similar mechanism of resistance. Virus levels were negatively correlated to transcript levels within the transgenic plants. This resistance will have practical applications for the control, of PLRV. Elucidation of the mechanism of resistance may also help understand the mechanisms of virus infection. / Land and Food Systems, Faculty of / Graduate

M.Sc. / As part of an investigation into differential gene expression in response to abiotic and chemical inducers of acquired resistance in tobacco, a PCR fragment of 660bp was repeatedly found in RNA preparations from treated cell suspensions by differential display analysis. The fragment (D1B) was isolated, purified, cloned and sequenced. The nucleotide sequence of the fragment was compared with sequences in the BLAST sequence database and was found to be homologous to the mitochondrial orfx genes from Arabidopsis thaliana, Beta vulgaris, Oenothera berteriana, Oryza sativa and Marchantia polymorpha. In order to obtain the full sequence of the gene specific primers were designed using the Arabidopsis sequence as template. The primers were designed to complete the 5’-end of the gene and were designed to overlap the D1B fragment previously found. A fragment (C3Y) of 460bp was isolated, purified, cloned and sequenced. The complete sequence (D1B and C3Y combined) was 851bp long and showed 96% homology with the Arabidopsis orfx gene on the nucleotide level and 87% homology on the translated amino acid level. The sequence was submitted to the Basic Local Alignment Search Tool (BLAST) database as accession gi: 24209907. In plant genomes, the orfx gene is closely linked to important structural genes such as the nad subunits of complex I (NADH: ubiquinone oxidoreductase). Orfx codes for a hypothetical protein that shows homology to the mttB (membrane targeting and translocation) gene found in E. coli. In bacteria the gene is essential because if deleted, the organism was no longer viable. Functional analysis of the bacterial gene revealed a novel pathway specific for membrane targeting and secretion of cofactor containing proteins, such as iron-sulphur (Fe-S) clusters, of which the mttB gene encodes one subunit. It is thought that a similar pathway might be responsible for the correct localisation and assembly of such Fe-S containing protein complexes in the inner mitochondrial membrane of higher plants. The differential display result may be indicative of a general up-regulation of mitochondrial gene expression in response to the triggering of plant defences or a possible specific effect on the expression of the orfx gene. A hypothesis was formulated that chemical inducers of plant defences affect the mitochondria of treated plant cells to result in increased production of reactive oxygen intermediates (ROI), similar to the oxidative microbursts proposed to be involved in systemic required resistance. Using a dichlorodihidrofluorescein (H2DCFDA) assay, it was found that salicylic acid (SA), benzo (1,2,3) thiadiazole-7-carbothioc acid S-methyl ester (BTH) and isonitrosoacetophenone (INAP) increased ROI production within cells in a dose dependant manner. The biochemical basis of this effect could possibly be related to the inhibition of the NADH:ubiquinone oxidoreductase activity of complex I of the mitochondrial electron transport chain by SA, BTH and INAP. / Prof. I.A. Dubery

M.Sc. / Polygalacturonase-inhibiting proteins (PGIPs) are cell wall-associated plant proteins that inhibit endopolygalacturonases from phytopathogenic fungi. It has been proposed that pgip encoding genes could be utilised for engineering increased resistance in transgenic crops against important fungal pathogens such as Botrytis cinerea. During this study a pgip gene from Malus domestica cv Granny Smith apple fruit was cloned by the degenerate and inverse polymerase chain reaction (PCR) techniques. An alignment of the pear and bean PGIP sequences was used to design degenerate PCR primers in highly conserved regions. Degenerate PCR allowed the amplification of a 351bp internal fragment of the pgip gene, termed ipgip. The DNA sequence of ipgip was used to design inverse PCR primers. A Southern blot of apple genomic DNA probed with the ipgip fragment was used to identify restriction enzyme sites for inverse PCR. Inverse PCR enabled cloning of the remainder of the gene, from which a composite pgip gene sequence was constructed. The composite apple pgip gene comprised an open reading frame of 990bp that is predicted to encode a 330 amino acid polypeptide. The polypeptide contains a putative 24 amino acid N-terminal leader sequence that may function as a signal peptide for secretion. The deduced apple PGIP contains nine cysteine residues and seven potential N-linked glycosylation sites. Ten loosely conserved leucine-rich repeat motifs characteristic of PG1Ps were identified in the apple PGIP sequence. The apple PGIP showed 97% and 55% amino acid identity to the pear and bean PGIPs, respectively. The full-length apple pgip gene was re-isolated from genomic DNA by PCR using primers designed to the 5' and 3' ends of the composite pgip gene. The apple pgip gene was cloned into a plant transformation vector and transformed into tobacco by Agrobacterium-mediated transformation. Phenotypically normal transgenic tobacco plants were produced. Stable transgene insertion into the transgenic tobacco genomes was verified by PCR and Southern blot analyses. Sequence analysis of the pgip construct used for transformation revealed two potential mutations in the deduced amino acid sequence. The substitutions of Asp residues with Asn and Tyr at positions 43 and 196, respectively, could interfere with the secondary structure of the expressed transgene protein. To test whether the apple PGIP was effective against Botrytis cinerea, protein extracts were prepared from apple fruit and transgenic tobacco and tested for inhibitory activity against B. cinerea polygalacturonases. Biochemical assays showed that a heat-denaturable PGIP extract prepared from apple fruit inhibited the polygalacturonases produced by a virulent isolate of Botrytis cinerea grown on pectin and apple cell walls. Protein extracts prepared from transgenic tobacco did not show any inhibitory activity towards Botrytis polygalacturonases. This suggests the absence of active PGIP in the extracts possibly due to inefficient transcription of the transgene or due to the introduced mutations.

X-disease, caused by phytoplasmas, is one of the destructive diseases in stone fruit trees, causing yield loss and poor fruit quality. So far no effective methods are available to control X-disease. X-disease resistance has been first discovered in chokecherry (Prunus virginiana, 2n=4x=32), which is a native woody species of North America. To identify molecular markers linked to X-disease resistance, simple sequence repeat (SSR) markers were used to construct genetic linkage maps for chokecherry and to identify markers associated with X-disease resistance in chokecherry. In this research, three segregating populations of chokecherry were developed by crossing one X-disease resistant (CL) with three susceptible chokecherry lines (a, c, and d), of which the progenies were 101, 177, and 82, respectively. In order to construct a genetic map for chokecherry, 108 pairs of SSR primers were employed from other Prunus species. Additionally, a set of 246 SSRs were developed from chokecherry sequencing by Roche 454 sequencing technology. A total of 354 pairs of SSR primers were used to screen individuals of all three populations. Two software programs, TetraploidMap and JoinMap, were used to construct linkage map based on single-dose restriction fragments (SDRFs) and two parental linkage maps were generated for each population from both software programs. Bulked segregant analysis (BSA) was applied for identification of X-disease resistance markers. As a result, one SSR marker was found to be linked to the X-disease resistance. The set of 246 chokecherry SSRs was later used to test transferability among another 11 rosaceous species (sour cherry, sweet cherry, wild cherry, peach, apricot, plum, apple, crabapple, pear, june berry, and raspberry). As a result, chokecherry SSR primers can be transferable in Prunus species or other rosaceous species. An average of 63.2% and 58.7% of amplifiable chokecherry primers amplified DNA from cherry and other Prunus species, respectively, while 47.2% of amplifiable chokecherry primers can be transferable to other rosaceous species. The genetic information, including genetic map, disease linked marker, chokecherry sequence, and confirmed transferability of the identified chokecherry SSRs to other species, will benefit the genetic research in Prunus and other rosaceous species.

Sclerotinia sclerotiorum causes white mold and severe yield losses of pea. 484 accessions from the Pisum core collection were screened for resistance using a mini-agar plug technique. 49, 41, and 13 accessions were identified with partial resistance based on lesion expansion inhibition (LEI), nodal transmission inhibition (NTI), and both traits combined, respectively. A genetic linkage map based on F2 DNA from the cross, Lifter/PI240515, was developed with 78 markers on 9 linkage groups (LG) spanning 734 cM. Two quantitative trait loci (QTL) were identified based on phenotypic data from F2:3 and F3:4 families. A single QTL on LGIII explained 34.1% of the phenotypic variation for LEI, while a second QTL on LGII(b) explained 2.5% of the phenotypic variation for NTI. This is the first report of QTL for S. sclerotiorum resistance in pea which will be useful in development of resistant pea varieties.

No description available.

No description available.

No description available.

No description available.