Massey Documents by Type

Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294

Browse

Filters

Settings

Subject: search.filters.subject.Plant molecular genetics

Search Results

Now showing 1 - 7 of 7
  • Thumbnail Image
    Item
    The detection of plasmid transfer genes in Rhizobium species : a thesis presented in partial fulfilment of the requirement for the degree Master of Science in Microbiology at Massey University, New Zealand
    (Massey University, 1994) Fisher, Paul Jamie
    In order that Rhizobium tra genes responsible for Sym plasmid transfer might be found, DNA probes were constructed from Agrobacterium tra genes. Three probes were constructed from DNA containing;- 1) traR, a gene which regulates other tra genes on the Agrobacterium tumefaciens plasmid pTiC58, 2) an OriT site, at which nickases cleave the plasmid before conjugal transfer can take place, and 3) part of a gene required for construction of a mating bridge. All three probes were constructed by the ligation of tra areas from the A.tumefaciens strain C58 plasmid pTiC58 into broad host range plasmid vectors and subsequent electroporation into E.coli cells. The genomic DNA digests of several Rhizobium and Agrobacterium strains were blotted and probed with the three probes under various washing and hybridisation stringencies. A.tumef aciens strain LMG64 was the only Agrobacterium strain aside from strain C58 to have DNA homologous to any of the probes. Neither R.leguminosarum bv trifolii strain ICMP2163, nor R. leguminosarum bv trifolii strain ICMP2163::Tn5, nor R.leguminosarum bv trifolii strain PN165 had any DNA homologous to any of the probes. However, R.leguminosarum bv trifolii strain ATCC14480 showed homology to the tra I probe (containing traR), and R.loti strain ATCC33669, phylogenetically the most distant relative to A.tumefaciens strain C58 shared homology with the tra III probe (containing DNA responsible for mating bridge assembly). Therefore the distribution of tra genes from the Ti plasmid of A.tumefaciens strain C58 among the agrobacteria and rhizobia used in this study did not correlate to their phylogenetic relatedness to A.tumefaciens strain C58 or to one another.
  • Thumbnail Image
    Item
    Characterisation of a Rhizobium loti nodulation mutant : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University
    (Massey University, 1991) Collins-Emerson, Julie Marie
    The aim of the project was to characterise the Rhizobiurn loti Nod- Tn5 mutant strain, PN233. The Tn5 insertion had been previously localised to a 7.1 kb Eco RI chromosomal fragment. This fragment was sub-cloned and a Bam HI/Sal I endonuclease restriction map for the region was determined. Hind III digests were utilised to identify the approximate location of the Tn5 233 insertion and those of four other Tn5 insertions (4016, 4019, 4047 and 4053) in the 7.1 kb region. The 233 mutation was found to map to a 1.45 kb Sal I fragment and that of an overlapping 2.8 kb Barn HI fragment. The 7.1 kb Eco RI fragment and a larger 22.7 kb fragment that encompassed this region, had been cloned into pLAFRl. The construct carrying the 22.7 kb fragment (pPN305) was crossed into four R.l. bv. trifolii strains, each mutant in one of the four common nod genes, A,B,C, and D. The construct was able to complement the nodC mutation indicating the presence of a nodC gene somewhere on the 22.7 kb region. The mutations 4047 and 4053 had been found to map to either side of the 233 Tn5 insertion. Both insertions affected nodule formation and were thus included in further plant complementation tests. These experiments involved crossing both the pPN305 and a construct bearing the smaller 7.1 kb Eco RI fragment (pPN25) into the R. loti and R.l. bv. trifolii Tn5 mutants. What was unusual about the results was that, while the 7.1 kb fragment was able to complement the mutations, the larger 22.7 kb fragment which encompasses that region could complement PN4047 and PN4053 but was unable to complement the PN233 mutant. The 2.8 kb Barn HI and 1.45 kb Sal I fragments, to which the 233 insertion was mapped, and that of an adjacent 1.2 kb Sal I fragment, were sub-cloned and then Bal 31 digested in both orientations to create a series of overlapping fragments. These fragments were then sequenced. The data revealed that the 233 Tn5 had inserted into the R. loti node gene. It was determined that the 4047 Tn5 was also located in this gene, slightly upstream of 233, while 4053 had inserted into the 5'-region of nodI which is downstream of nodC. Nod.A was identified upstream of nodC indicating an arrangement of common nod genes different from the conventional nod.ABeIJ found in other rhizobia. The promoter for these nod genes, the nod box, was located upstream of the nodA gene. A particularly puzzling aspect of the results is that, while PN4047 is complemented by both pPN305 and pPN25, PN233, which has an insertion in the same gene, could only be complemented by the smaller fragment carried by the pPN25 construct. To explain this result, it is proposed that PN233 is producing a mutant NodC protein and that this, in combination with doubled copies of a gene or genes present elsewhere on the 22.7 kb fragment, is responsible for interfering with complementation in this mutant. Alternatively, it may be that the imbalance of doubled copies of downstream, co-transcribed genes in the presence of one copy of a functional node gene causes complementation failure.
  • Thumbnail Image
    Item
    Functional characterisation of constitutive expresser of pathogenesis-related genes 5 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2017) Faisal, Muhammad
    As reported previously, CPR5 negatively regulates the onset of leaf death, hypersensitive response, disease resistance and early leaf senescence. cpr5 plants contain aberrant trichomes and higher levels of ROS, SA and JA. Cell-cycle, JA/ET, ABA and sugar signalling are also affected in cpr5 plants. These results suggest that CPR5 is a master regulator of multiple processes. However, how CPR5 manages to exert pleiotropic effects is still poorly understood. The first objective of the current study was the purification of the CPR5 protein to solve its crystal structure. Extensive in silico analyses were carried out and the results showed that CPR5 is predicted to be a membrane protein with 4 or 5 transmembrane (TM) domains. Additionally, CPR5 contains intrinsically disordered regions (IDRs) at its N-terminus. Proteins containing IDRs and TM domains are often difficult to purify for crystallization studies. Therefore, the undesirable regions of CPR5 such as, IDR and TM domains were deleted and a set of 24 constructs were developed. Despite several efforts, none of the CPR5 recombinant proteins were isolated. In addition to predicting IDR and TM domains, in silico results also predicted three NLS-encoding clusters, casein kinase phosphorylation sites, multiple start codons, coiled-coil domains and glycine motifs. To find out the roles of these putative structural elements on CPR5 functions, firstly a CPR5 cDNA was synthesised and termed as SynCPR5. Subsequently, predicted sites or motifs were mutated in SynCPR5 through sitedirected mutagenesis and a set of 25 mutated CPR5 transgenes (cDNA constructs) were developed. Using a complementation strategy, all the constructs were transformed into cpr5- 2 plants. The results show that the complementation of cpr5-2 plants with SynCPR5, fully restored HR-like lesions, wildtype-like trichomes and leaves on SynCPR5 plants. Further physiological characterization such as, transcript abundance of SynCPR5, PR1, PR5 and PDF1.2, leaf area measurements and ploidy levels showed that CPR5 regulates some of its functions and phenotypes quantitatively as well as qualitatively. When compared with the wildtype, better growth (larger leaves) but enhanced disease susceptibility was found in metCPR5 transgenic lines (in which putative start codons were mutated), indicating that CPR5 regulates a balance between growth and resistance. Functional characterization of NLS mutants (nlsCPR5) showed that NLS-encoding clusters are important for CPR5 proper functions. However, current evidence is insufficient to relate their role in CPR5 localization. Moreover, in silico results show that putative NLS clusters are present in the region of CPR5 which were annotated as intrinsically disordered region (IDR). Similar phenotypes shown by both nlsCPR5 and Del63CPR5 (in which the first 63 amino acids of CPR5 including putative NLS were deleted), indicate that the putative NLS clusters could be part of IDR and may have dual functions. Loss-of-function phenotypes shown by coiled-coil domain mutants (ccdCPR5) reinforce the role of coiled-coil domains in CPR5 homo-dimerization. Moreover, in contrast to previous reports, the downregulation of PDF1.2 in the majority of CPR5 complementation lines proposes CPR5 to be a positive regulator of PDF1.2. Based on the results presented in the current study, putative CPR5 IDRs and coiled-coil domains are proposed to facilitate CPR5 dimerization in order to restrict the entry of deregulated cargos into the nucleus. Moreover, these results uncover a novel role of CPR5 in the regulation of balance between plant growth and resistance. Furthermore, this study, for the first time, reports evidence of the requirement of NLS clusters for CPR5 functions.
  • Thumbnail Image
    Item
    Molecular characterisation of the HMG CoA reductase gene from Neotyphodium Lolii : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 1997) Dobson, Joanne Margaret
    3-Hydroxy-3-methylglutaryl Coenzyme A reductase (Hmg) catalyses the conversion of HMG CoA to mevalonic acid: the first step of the isoprenoid biosynthetic pathway. This pathway produces a wide variety of primary metabolites which are involved in many different cellular processes. Neotyphodium endophytes in association with the grass host are known to produce a range of secondary metabolites including the indole diterpenoids (eg paxilline and lolitrem) and the ergopeptine alkaloids (eg ergovaline). Given that these pathways are upregulated in planta the availability of mevalonic acid, be it from fungal or plant source, will be important in controlling the levels of the different toxins synthesised. The aim of this work was to clone the fungal endophyte hmg and characterise the promoter to enable study of its regulation in planta via reporter gene studies. Using degenerate primers designed against conserved regions of other hmg genes a 359 bp fragment was amplified from the Neotyphodium lolii isolate Lpl9, which grows in perennial ryegrass (Lolium perenne). DNA sequencing confirmed that the sequence amplified was part of a unique hmg gene. Southern hybridisations suggest that there is a single copy of hmg in strain Lpl9 (a haploid) but two copies in strain Lpl (an interspecific hybrid; Schardl et al. 1994). The fragment of N. lolii hmg was used to screen a λGEM-12 genomic library of Lpl9 and four positive overlapping clones were isolated. Fragments of one clone. λJD12, were subcloned, sequenced and a physical map of this region of the genome was constructed. The entire sequence of hmg was determined using primer walking and was found to encode a 1188 amino acid polypeptide. From comparison to other Hmg proteins the catalytic domain has been shown to be highly conserved while the amino-terminal domain, containing transmembrane regions is divergent with very little sequence similarity near the translation start site and promoter region. Using RT-PCR analysis the hmg gene was shown to consist of two open reading frames separated by a 73 bp intron. RT-PCR was also used to determine the location of the transcriptional start site and this is supported by the presence of putative CAAT and TATA consensus sequences. With the promoter region identified and characterised further analysis of the regulation of hmg in planta can be undertaken.
  • Thumbnail Image
    Item
    Characterization of two genes involved in Neotyphodium lolii growth : a thesis presented in partial fulfillment of the requirements for the degree of MAster of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2004) McMillan, Duncan George Glenn
    Neotyphodium lolii is a filamentous fungus that forms symbiotic associations with Lolium perenne, growing in its intercellular spaces. It is a feature of the symbiosis that growth of the fungus and the plant is synchronized. When the grass leaf-blade grows, the fungus grows at the same rate, hence when the blade ceases extension the hyphae do likewise. In addition, in planta there is little hyphal branching, where as in culture hyphae branch at regular intervals. This suggests the existence of a regulatory mechanism in planta that partially dictates hyphal morphology and growth. The criteria for choosing possible candidate genes relied on whether the gene had a function relating to hyphal branching and/or regulation of hyphal extension in several organisms. Three candidate genes were selected. Protein elongation factor 2 (EF-2; an elongation factor associated with the ribosome) was targeted to add more direct evidence to the high metabolic rate observed in planta using the GUS reporter gene by Tan et al (2001). Cell division control protein 12 (CDC-12); a septin which is involved in the construction of the 10 nm ring structure associated with cell division and whose mutation is lethal in yeast was chosen to help distinguish the growth mode of N.lolii in planta. A Stretch-activated Calcium Channel (SACC) which allows exogenous calcium into the cell upon application of lateral pressure on the membrane was targeted to help distinguish the possible recognition signal the hyphae make to elucidate when the host tissue is growing. This project was then divided into four parts, one part per gene and a final part looking at the in vitro and in vivo expression of these genes. For the first three parts degenerate PCR was performed and appropriate-sized fragments cloned, sequenced and restriction mapped for EF-2 and CDC-12 (2066 bp and 514 bp respectively). Database searches were used to identify the sequences as potentially being the target genes. Degenerate PCR was unsuccessful for the SACC. Southern blots were used to identify restriction enzymes for Inverse PCR; and this was used to obtain the remaining 5' and 3' regions of each target gene. Gene prediction software was used to predict gene structure; 5' and 3' RACE to confirm the length, introns and start/stop points of EF-2 and CDC-12 full gene transcripts (2,900 and 1,612 bp respectively). Internet-based sequence analysis tools subsequently were used to identify sequence features. For the second part, expression of EF-2 and CDC-12 are investigated during various states of hyphal growth. Growth curves were constructed and in vitro expression analysis was achieved by Northern blot. The expression patterns of EF-2 and CDC-12 followed the growth state of N.lolii. RT PCR was used to confirm in planta expression of both genes and validate their uses for future studies.
  • Thumbnail Image
    Item
    Cloning and characterisation of two subtilisin-like protease genes from Neotyphodium lolii : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Genetics at Massey University
    (Massey University, 2000) McGill, Michelle Kay
    PCR amplification of Neotyphodium lolii genomic DNA with degenerate primers detected two different sequences with homology to subtilisin-like proteases. These two PCR products were used to screen a N. lolii Lp19 genomic library. The prt1 gene was isolated by screening the genomic library with the GH30 PCR product. This gene encodes a putative peptide of 434 amino acids that is most similar to subtilisin-like proteases from Aspergillus sp. The prt1 gene contained a single intron, which was in a position conserved with other fungal genes. 3'RACE was used to determine the polyadenylation site for the prt1 gene. Repetitive DNA was a feature of both the 3' untranslated region (UTR) and sequences downstream of the prtl gene. Within the 3' UTR, a complex microsatellite was found extending over 50 base pairs. Downstream of the gene, a minisatellite locus of 360 base pairs in size was found, consisting of 40 copies of a 9 base pair AT-rich repeat. Expression of prt1 was examined in cultures with various types of carbon and nitrogen sources. Although no conclusive results could be drawn, the type of carbon and nitrogen available did have some effect on prt1 expression. Repression of prt1 expression was only observed in media supplemented with sucrose and glutamate. A 500 bp fragment from the prt1 promoter was introduced into the vector pFunGus to create a translational fusion with gusA. This vector, pMM9, was transformed into Penicillium paxilli. Although transformation frequencies were low, the transformants obtained appeared to be stable for hygromycin resistance. Expression of GUS was observed in seven out of twelve of the stable transformants. This showed that the promoter fragment in pMM9 was sufficient for expression of GUS in a heterologous system. The prt2 gene was isolated by screening a genomic library with the GH3 PCR product. Partial sequence has been obtained for the prt2 gene. The prt2 gene contains at least three introns, the first of which is conserved with prt1. From the sequence obtained, prt2 encodes a peptide with strong similarity to subtilisin-like proteases from Metarhizium anisopliae, a fungal pathogen of insects.
  • Thumbnail Image
    Item
    The indole-diterpene gene cluster from the ryegrass endophyte, Neotyphodium lolii, is required for the biosynthesis of lolitrem B, a bioprotective alkaloid : this thesis is presented as a partial fulfillment of the requirements for the degree of Doctor of Philosophy (Ph. D.) in Molecular Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2005) Young, Carolyn Anne
    Lolitrems are indole-diterpene alkaloids produced by Epichloë and Neotyphodium endophytes in association with their host grass Lolium perenne. Some indole-diterpene (ID) alkaloids are proposed to have insecticidal properties, but lolitrem B is known as the causative agent of the animal syndrome ryegrass staggers. Lolitrems are preferentially synthesised in planta. which suggests that the genes required for lolitrem biosynthesis are symbiotically expressed. The lolitrem biosynthesis pathway has been proposed as a metabolic grid based on the identification of likely intermediates from endophyte-infected ryegrass. Closely related ID compounds are expected to serve as substrates for the same enzyme, but until recently these steps had not been validated. The identification and characterisation of a Petticillium paxilli gene cluster required for the synthesis of the ID paxilline has identified key enzymes required for the production of the ID backbone. Based on the similarity of lolitrem B to paxilline it was proposed that these two biosynthesis pathways would share orthologous early steps but later steps to convert paxilline to the more complex lolitrem B would require additional enzymes. The lolitrem biosynthesis genes (ltm) were isolated using degenerate PCR and from candidate genes identified as ESTs in cDNA libraries. Ten ltm genes were identified that had functions consistent with those required for lolitrem B biosynthesis. The 10 ltm genes were contained on three gene clusters that are separated by repetitive AT-rich sequences that contain remnants of retrotransposons. The ltm clusters 1 and 2 contain eight genes, seven of which are orthologues of the characterised P. paxilli paxilline biosynthesis gene cluster (pax). Functional characterisation of ltmM an FAD-dependent monooxygenase and ltmC a prenyl transferase confirmed these two genes were required for ID biosynthesis and were orthologues of paxM and paxC, respectively. All 10 ltm genes have similar expression profiles and were highly expressed in planta where the production of lolitrem B is most prevalent. The taxonomic distribution of the ltm genes has established which endophyte strains are likely to produce ID compounds. This work provides the basis for elucidation of the lolitrem biochemical pathway and opens the way for determining how the plant regulates the synthesis of this important group of bioprotective molecules.