Browsing by Author "Faisal, Muhammad"

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    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.