Tobacco SABP2, a 29. kDa protein catalyzes the conversion of methyl salicylic acid (MeSA) into salicylic acid (SA) to induce SAR. Pretreatment of plants with acibenzolar-. S-methyl (ASM), a functional analog of salicylic acid induces systemic acquired resistance (SAR). Data presented in this paper suggest that SABP2 catalyzes the conversion of ASM into acibenzolar to induce SAR. Transgenic SABP2-silenced tobacco plants when treated with ASM, fail to express PR-1 proteins and do not induce robust SAR expression. When treated with acibenzolar, full SAR is induced in SABP2-silenced plants. These results show that functional SABP2 is required for ASM-mediated induction of resistance.

Tobacco SABP2 has been shown to display high affinity for salicylic acid (SA) and methylsalicylate (MeSA) and plays an important role in SAR signal development. Using biochemical approach, SABP2 has been shown to demonstrate strong esterase activity in converting MeSA to SA. Recent study shows that tetra fluoroacetophenone, a synthetic analog of SA, competitively inhibits SABP2 esterase activity as well as suppresses SAR signal development in tobacco mosaic virus (TMV)-infected tobacco plants. Not much has been studied on the effect of pesticides on plant defenses. Because both AChE and SABP2 are esterase-like proteins belonging to α/β hydroxylase superfamily, we hypothesize that pesticides may inhibit the MeSA esterase activity of SABP2 and block SAR development. Biochemical and molecular biology techniques were used to test this hypothesis. SAR in tobacco-TMV plant-pathogen system is measured by significant decrease in TMV-induced lesion sizes in secondarily inoculated distal leaves.

Thesis (M.Sc. (Horticulture)) -- University of Limpopo, 2022 / The South African Avocado Industry has recently announced plans to expand exports into new markets, such as the United States (US). As a requirement for these markets, fruit of high quality must be stored at ultra-low temperature to mitigate phytosanitary risks. However, ‘Hass’ avocado fruit are susceptible to chilling injury when stored at temperatures below 3°C. Moreover, CI development resulted in uneven ripening and disease infestation due to damaged cell membranes. Therefore, the objective of this study was to evaluate the potential of methyl jasmonate (MeJA) and salicylic acid (SA) on quality maintenance of 'Hass' avocado fruit during ultra-low cold storage. Matured ‘Hass’ avocado fruit were harvested at commercial dry matter (22%). The experiment was conducted using a completely randomized design (CRD) with eight replications per treatment. Treatment concentrations for methyl jasmonate (MeJA) were 0 (control), 10 and 100 μmol•L−1 , while those for salicylic acid (SA) were 0 (control), 1.0, 2.0 and 3.0 mM. After treatments, fruit were stored at 2°C for 31 days and thereafter, ripened at ambient temperature (±25°C) until fully ripe. During ripening, fruit were evaluated for weight loss, exocarp colour, firmness, chilling injury, as well as physiological (vascular browning) and pathological disorders (fruit rot). In this study, dipping fruit in MeJA solution significantly (P < 0.05) reduced ‘Hass’ avocado fruit firmness loss. Moreover, MeJA showed a significant effect (P < 0.05) on hue angle (h°) but did not significantly affect (P > 0.05) visual colour rating, chroma (C*), lightness (L*) and weight loss. The results showed that ‘Hass’ avocado fruit treated with 10 μmol•L−1 MeJA reduced weight loss when compared with 100 μmol•L−1 MeJA from day 2 to day 8 of ripening. Overall results showed a visual change in ‘Hass’ avocado fruit exocarp colour, with eye colour changing from rating 1 (emerald-green) to 3 (olive- green) for control and fruit treated with MeJA throughout the ripening days. Furthermore, MeJA reduced ‘Hass’ avocado fruit external chilling injury, physiological and pathological disorders. With respect to SA treatments, the result showed that dipping fruit at 1.0 and 2.0 mM SA had a significant effect (P < 0.05) on reducing firmness loss during ripening. Salicylic acid (1.0 mM) reduced and alleviated ‘Hass’ avocado fruit external chilling injury during ultra-low cold storage. Furthermore, result showed that 1.0 and 2.0 mM SA treatments had significant affect (P < 0.05) on firmness loss. Moreover, a significant effect was observed on visual colour and C* but did not affect (P > 0.05) L* and h°. Fruit treated with SA showed poor exocarp colour development with extended exposure to ultra-low cold storage, as a result, developed chilling symptoms. The treatment of ‘Hass’ avocado fruit with 1.0 mM SA inhibited the incidence of fruit rot and vascular browning when compared with control and fruit treated with 2.0 and 3.0 mM SA. In conclusion, 10 and 100 μmol•L−1 MeJA and 1.0, 2.0 and 3.0 mM SA effectively preserved ‘Hass’ avocado fruit quality during storage at ultra-low temperature. / Agricultural Sector Education Training Authority (AgriSeta) and National Research Foundation (NRF)

Salicylic Acid Binding Protein 2 (SABP2) is a 29kDa protein present in extremely low amounts in tobacco leaves. SABP2 processes the mobile defense signal, methyl salicylic acid generated in plants resisting microbial infection. The precise localization of SABP2 in plants is not known. SABP2 has not been shown to have any targeting signal peptides. This study was designed to determine localization of SABP2 in tobacco plants. Biochemical and immunological studies using antibodies against SABP2 suggest that it is localized to the chloroplast, associating with chloroplast envelope membranes. Chloroplast import assays confirm that SABP2 is associated with the chloroplast envelope membrane. Solubilization and analysis of chloroplast membrane proteins show that imported SABP2 associates with the chloroplast envelope membrane by weak hydrophobic and/or ionic interactions. Cellular localization and understanding mechanisms of SABP2 import to the chloroplast will be important from a metabolic engineering standpoint to enhance plant natural defense against microbial pathogens.

Salicylic acid binding protein 2 (SABP2) is one of the key enzymes in salicylic acid-dependent plant defense pathway. SABP2 is a 29 kDa protein present in extremely low abundance in plants and it catalyzes the conversion of signaling molecule methyl salicylate into salicylic acid. Although it has been shown that 6x His-tagged SABP2 over expressed in E. coli is a homodimer, its exact conformation in planta is still unknown. Therefore, we proposed to determine if SABP2 exist as a dimer and/or monomer under natural condition. To verify the exact conformation of native SABP2 protein in plant, SABP2 was purified from wild type tobacco using a 5-step purification protocol. Analysis of purified SABP2 in gel filtration and immunoblot assay suggested that SABP2 exists as a monomer in tobacco plant. Studies on SABP2 conformation will give us insight into the structure and functional relationship of this protein in salicylic acid-dependent disease resistance pathway.

SABP2 (Salicylic Acid Binding Protein 2) plays a vital role in the salicylic acid signaling pathway of plants both regarding basal resistance and systemic acquired resistance against pathogen infection. SIP-428 (SABP2 Interacting Protein-428) is a Silent information regulator 2 (SIR2) like deacetylase enzyme that physically interacts with SABP2 in a yeast two-hybrid interaction and confirmed independently by a GST pull-down assay. We demonstrated that SIP- 428 is an NAD+ dependent SIR2 deacetylase enzyme. Transgenic tobacco plants silenced in SIP- 428 expression via RNAi showed enhanced basal resistance to microbial pathogens. Moreover, these SIP-428-silenced lines also exhibited a robust induction of systemic acquired resistance. In contrast, the transgenic tobacco lines overexpressing SIP-428 showed compromised basal resistance and failed to induce systemic acquired resistance. These results indicate that SIP-428 is likely a negative regulator of SA-mediated plant immunity. Experiments using a SABP2 inhibitor showed that SIP-428 likely functions upstream of SABP2 in the salicylic acid signaling pathway. It also indicates that SABP2 is dependent on SIP-428 for its role in the SA signaling pathway. Subcellular localization studies using confocal microscopy and subcellular fractionation showed that SIP-428 localized in the mitochondria. These results clearly show a role for SIP-428 in plant immunity.

<p>Salicylic acid (SA) is a central plant defense signal. It is not only required for closing the stomata upon infection to prevent pathogens from entering into the plant apoplast, but also mediates defense responses activated by pathogen-originated microbe-associated molecular patterns (MAMPs) and effectors in the infected tissues. In addition, SA is a necessary and sufficient signal for systemic acquired resistance (SAR). In <italic>Arabidopsis</italic> <italic>thaliana</italic>, SA level increases in response to pathogen attack, which is essential for activating defense responses. This SA accumulation involves transcriptional activation of several genes including <italic>ICS1</italic> (<italic>ISOCHORISMATE</italic> <italic>SYNTHASE</italic> <italic>1</italic>), <italic>EDS5</italic> (<italic>ENHANCED</italic> <italic>DISEASE</italic> <italic>SUSCEPTIBILITY</italic> <italic>5</italic>), <italic>EDS1</italic> (<italic>ENHANCED</italic> <italic>DISEASE</italic> <italic>SUSCEPTIBILITY</italic> <italic>1</italic>), <italic>PAD4</italic> (<italic>PHYTOALEXIN-DEFICIENT</italic> <italic>4</italic>) and <italic>PBS3</italic> (<italic>avrPphB</italic> <italic>SUSCEPTIBLE</italic> <italic>3</italic>). However, it is not well understood how pathogenic signals induce these SA accumulation genes. Interestingly, our time-course transcriptome analysis showed that these five genes share a similar pathogen-induced expression pattern, suggesting the existence of common transcription factors (TFs). Through yeast-one-hybrid screening, a TF NTL9 was identified for its interactions with the promoters of the SA accumulation genes. Preferentially expressed in guard cells, NTL9 activates the expression of SA accumulation genes in guard cells. The <italic>ntl9</italic> mutant is defective in pathogen-induced stomatal closure mediated by a well-characterized MAMP, flg22. Consistent with the stomatal closure defect, the <italic>ntl9</italic> mutant exhibits elevated susceptibility to surface-inoculated pathogens. The stomatal closure defect of the <italic>ntl9</italic> mutant can be rescued by exogenous application of SA, demonstrating that NTL9 acts upstream of SA in stomatal closure response. These results suggest that NTL9-mediated activation of SA accumulation genes is essential for MAMP-triggered stomatal closure.</p><p>While plants induce SA to activate defense responses, pathogens can also produce virulence factors to counteract the effects of SA. Coronatine is one such virulence factor produced by <italic>Pseudomonas</italic> <italic>syringae</italic>. Coronatine is known to promote opening of stomata for bacterial entry, bacterial growth in the apoplast, systemic susceptibility and development of disease symptoms such as chlorosis. In the process of examining the mechanisms underlying coronatine-mediated virulence, three homologous TFs, ANAC019, ANAC055 and ANAC072, were found to be activated by coronatine directly through the TF, MYC2. Genetic characterization of these three TF mutants revealed that these TFs mediate multiple virulence effects of coronatine by inhibiting SA accumulation. To exert this inhibitory effect, these TFs repress <italic>ICS1</italic> and activate <italic>BSMT1</italic>, genes involved in SA biosynthesis and inactivation modification, respectively. Thus, a signaling cascade downstream of coronatine was illustrated to dampen SA-mediated defense responses through differential transcriptional regulation of genes related to SA level.</p><p>Taken together, my dissertation studies revealed novel transcriptional regulation of SA production and demonstrated that this transcriptional regulation is a vital point not only for plant defense activation but also for pathogen manipulation to counteract defense responses. Further studies on the interplay of this transcriptional regulation by different TFs would broaden our understanding about the dynamics of plant-pathogen interaction.</p> / Dissertation

<p>Plants have evolved the circadian clock to anticipate environmental changes and coordinate internal biological processes. Recent studies unveiled the circadian regulation on plant immune responses as well as a reciprocal effect of immune activation on the clock activity. However, it is still largely unknown how the circadian clock interacts with specific immune signals. Plant hormone salicylic acid (SA) is a key immune signal. Its accumulation is sufficient to trigger immune responses and establish broad-spectrum resistance, known as systemic acquired resistance (SAR). My dissertation work studied whether SA could interact with the circadian clock and what potential mechanisms and the biological significance are.</p><p>I first found that SA could reinforce the circadian clock through the modulation of redox state in an NONEXPRESSER OF PR 1 (NPR1)-dependent manner. The basal redox state manifested by the NADPH abundance is shown to display a circadian rhythm. Perturbation in this cellular redox rhythm caused by the immune signal SA is sensed by the master immune regulator NPR1. NPR1 then triggers defense genes expression to generate SAR as well as transcriptionally activates several clock genes to reinforce the circadian clock. Since the basal redox state, which reflects the cellular metabolic activities, is under the circadian control, the reinforced circadian clock may negate the SA-triggered redox perturbation to restore the normal redox rhythm. One of NPR1-regulated clock components is TIMMING OF CAB2 EXPRESSION 1 (TOC1). SA/NPR1-mediated increase in TOC1 expression alone could lead to dampening of SAR through direct transcriptional repression on defense genes. Since maintenance of the immune responses is an energy-costly process, the strength and duration of SAR, a preventative defense strategy, need to be fine-tuned to reduce unnecessary energy expenditure. Therefore, both SA-dependent circadian clock reinforcement and the specific clock component TOC1 induction help to ensure a proper immune induction and a balanced energy allocation between defense and normal metabolic activities.</p><p>Besides the SA effects on the circadian clock, the circadian clock is found to reciprocally regulate SA biosynthesis. The clock gene, CCA1 HIKING EXPEDITION (CHE), and the major SA synthesis gene, ISOCHORISMATE SYNTHASE 1 (ICS1), show in-phase oscillatory rhythms, indicating that CHE may contribute to generation of the circadian rhythm of the basal SA level. I found that CHE, as a transcription factor, directly binds to the promoter of ICS1 to positively regulate its expression. After pathogen infection, CHE promotes endogenous SA biosynthesis and acts as a positive regulator of SAR. The function of the clock component CHE in activating ICS1 not only reveals a novel transcriptional regulatory mechanism of SA accumulation but also provides a new molecular link between the circadian clock and plant immunity.</p><p>In summary, my dissertation studies identified previously unknown molecular mechanisms of how the circadian clock mediates SA biosynthesis and SA-triggered immune responses. The interplay between the circadian clock and SA achieves a balance between activation of immune responses and maintenance of normal metabolic activities. Further studies may explore how other plant immune signals affect the circadian clock as well as how different clock components coordinately regulate the plant immunity. These future directions will broaden our understanding about the clock-immunity crosstalk.</p> / Dissertation

The base excision repair protein, MBD4 (also known as MED1) is known to be transcriptionally coupled to a mismatch repair protein MLH1. To date the significance of this coupling has not been elucidated and the significance of MBD4 within the mismatch repair system and apoptotic pathway is still being understood. Recently a novel alternatively spliced form of MBD4 has been identified and termed sMBD4. To date the significance of sMBD4 is unknown. MBD4 and sMBD4 share a common glycosylase domain and this is the domain through which MBD4 is reported to interact with MLH1. It was the aim of this study to determine if sMBD4 was also a binding partner of MLH1 to help elucidate a potential role of sMBD4 and to further characterise the binding domain between MLH1 and MBD4. Recombinant proteins were utilised in binding assays however, a specific protein – protein interaction could not be determined. Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of DNA mismatch repair (MMR) proteins in DNA MMR proficient cells. As MBD4 has previously been suggested to be coupled to MLH1 expression by a post‐translational mechanism the cytotoxicy of aspirin in relation to MBD4 expression was examined. This study reports that aspirin does not up‐regulate MBD4 gene transcription in vitro in the DNA mismatch repair proficient/p53 mutant colorectal cancer cell line SW480. However, MBD4 gene transcription was up‐regulated upon treatment with the aspirin precursor, salicylic acid. The suggested involvement of the DNA repair proteins in the mechanism of action of aspirin promoted the investigation into the expression of DNA damage signalling pathways genes upon aspirin exposure. This study utilised a commercially available PCR array to analyse the expression of 84 DNA damage signalling genes in the SW480 colorectal cancer cell line upon aspirin treatment. It is reported that treatment of the SW480 cell line with aspirin caused changes in mRNA expression of several key genes involved in DNA damage signalling including a significant down‐regulation in expression of the genes encoding ATR, BRCA1 and MAPK12 and increases in the expression of XRCC3 and GADD45α genes. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo‐protective effect of aspirin in vivo.Further to this, protein expression was analysed to determine if correlation could be established with the changes in mRNA expression observed. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45α, an increase in XRCC3 protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This study indicates that alterations in gene expression seen in microarray studies need to be verified at the protein level. Furthermore, this study reports the novel discovery of XRCC3 gene and protein expression being susceptible to exposure to the non‐steroidal anti‐inflammatory drug, aspirin.

Infection of the resistant Arabidopsis ecotype Di-17 with Turnip Crinkle Virus (TCV) elicits hypersensitive response (HR), accompanied by increased expression of defense genes. HR to TCV is conferred by HRT, which encodes a coiled-coil (CC)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR) class of resistance (R) protein. In contrast to HR, resistance requires HRT and a recessive locus designated rrt. Unlike most CC-NBS-LRR R proteins, HRT-mediated resistance is dependent on EDS1 and independent of NDR1. Resistance is also dependent on salicylic acid (SA) pathway and light. A dark treatment, immediately following TCV inoculation, suppresses HR, resistance and activation of a majority of the TCV-induced genes. To determine the genetic, molecular and biochemical basis of light-dependent defense pathway, we studied the role of various photoreceptors in HRT-mediated resistance to TCV, HRT protein levels and its localization. Interestingly, mutation in blue-light photoreceptors led to degradation of HRT via a proteasome-dependent pathway and resulted in susceptibility to TCV. Exogenous application of SA induced transcription of HRT, which restored HRT levels in some, but not all, mutant backgrounds. These results show that different photoreceptors function distinctly in maintaining post-transcriptional stability of HRT. In addition to photoreceptors, HRT also forms a complex with several other proteins, many of which participate in the RNA silencing pathway and are required for HRT-mediated resistance. Together, our results suggest that HRT forms a multi-protein complex and that HRT-mediated signaling involves reconstitution of this complex.