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Item Identification of blueberry leaf rust pathogen and quantification of disease infection levels in a blueberry plantation in Hastings, NZ : a thesis presented in partial fulfilment of the requirements for the degree of Master of AgriScience in Horticulture at Massey University, Turitea Campus, Palmerston North, New Zealand(Massey University, 2019) Chen, XiaoyingBlueberries (Vaccinium spp.) are a favourite fruit and they are produced worldwide. In New Zealand, blueberries are the main export berry fruit and contribute greatly to export income. More than 2,800 tonnes of blueberries were produced in the 2017/2018 harvesting season and earned $34.8 million export income in 2018. Currently, 740 ha of the blueberry plantations can be found in both the South Island and North Island. Otautau is the main growing region in the South Island while Waihopo, the Waikato regions of Ngatea and Ohaupo, the Bay of Plenty and Hastings are the regions in the North Island, producing most of the fresh blueberries in New Zealand. However, blueberry leaf rust has become a prevalent disease in blueberry production and a concerning issue for blueberry growers. In Hastings production sites, serious infections have been found in recent years. Although fungicides were applied to control blueberry leaf rust, this form of control is incomplete and unsustainable for blueberry production. The deployment of varieties that are naturally resistant would be a better option for managing blueberry leaf rust disease. Currently, few cultivars are available for this purpose, but breeding for rust resistance can address this demand. The main issues preventing the production of resistant varieties are insufficient knowledge about this rust pathogen in New Zealand, and the lack of resistant germplasm sources and efficient resistance screening procedures. In this study, using the morphological characteristics and genome sequencing results based on the Internal Transcribed Spacer (ITS) regions, Thekospora minima was identified as the causal organism of blueberry leaf rust disease in Hastings, Hawke’s Bay, New Zealand. Additionally, a field assessment was used for understanding the blueberry rust disease resistance levels in current blueberry cultivars. The disease incidence and disease severity of 23 blueberry cultivars, including five rabbiteye, three northern highbush and fifteen southern highbush, were assessed using Fiji software during the 2019 harvesting season. Based on a Tukey Honest Significant Differences (TukeyHSD) analysis, these observed blueberry cultivars were divided into four infection levels of blueberry leaf rust using the percentage of the infected area on the leaf (PIAL). ‘Scintilla’ was highly susceptible to blueberry leaf rust disease, while ‘Blue Moon’ and ‘Southern Splendour’ were moderately susceptible. Nineteen blueberry cultivars, made up of ‘Rahi’, ‘Centra Blue’, ‘Centurion’, ‘Titan’, ‘Sky Blue’, ‘Nui’, ‘Duke’, ‘Camellia’, ‘Misty’, ‘Springhigh’, ‘Snowchaser’, ‘Miss Jackie’, ‘Miss Lily’, ‘Georgia Dawn’, ‘Suziblue’, ‘Kestrel’, ‘Flicker’, ‘Sweetcrisp’ and ‘Palmetto’, showed susceptibility to this rust disease, and ‘‘O’ Neal’ was the one that showed partial resistance to the blueberry rust infection. Furthermore, using 1.5×104 concentration inoculum, an inoculation test was completed in a temperature-controlled room at the Plant Growth Unit of Massey University. The inoculum was sprayed on the healthy leaves from detached branches of the ‘Sky Blue’ blueberry cultivar and they were grown in reverse osmosis water for a 35-day observation on rust symptom development. Fiji software was applied in the assessment of disease severity in this inoculation test. A strong correlation (>0.99) was found between the increase in lesion area (ILA) from the inoculation test and the PIAL from field assessment. A preliminary prediction equation was established by a simple linear regression model. This equation can be used to predict the blueberry leaf rust level on different blueberry cultivars and breeding materials under field conditions by using the results from an inoculation test. This model would be an efficient approach for assisting the screening on blueberry leaf rust of blueberries.Item Identification of mechanisms defining resistance and susceptibility of Camellia plants to necrotrophic petal blight disease : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Manawatū, New Zealand(Massey University, 2019) Kondratev, NikolaiSpecies in the genus Camellia, which includes the tea crops, oil-producers and valuable ornamental plants, have economic and cultural significance for many countries. The fungus Ciborinia camelliae causes petal blight disease of Camellia plants, which has a short initial asymptomatic phase and results in rapid necrosis and fall of blooms. Ciborinia camelliae is a necrotrophic pathogen of the family Sclerotiniaceae, which also includes two broad-host range necrotrophic pathogens, Botrytis cinerea and Sclerotinia sclerotiorum. Previously it was shown that some Camellia plants, such as Camellia lutchuensis, are naturally resistant to petal blight. In order to find molecular mechanisms underpinning this resistance, a genome-wide analysis of gene expression in C. lutchuensis petals was conducted. The analysis revealed a fast modulation of host transcriptional activity in response to C. camelliae ascospores. Interaction network analysis of fungus-responsive genes showed that petal blight resistance includes increased expression of important plant defence pathways, such as WRKY33-MPK3, phenylpropanoid and jasmonate biosynthesis. A much-delayed activation of the same pathways was observed in the susceptible Camellia cultivar, Camellia ‘Nicky Crisp’ (Camellia japonica x Camellia pitardii var. pitardii), suggesting that failure to activate early defence enables C. camelliae to invade and cause tissue necrosis. Early artificial induction of defence pathways using methyl jasmonate reduced the rate of petal blight in susceptible ‘Nicky Crisp’ plants, further verifying the role of a rapid defence activation in petal-blight resistance. Overall, transcriptomic and functional analysis of the Camellia spp.- C. camelliae interaction demonstrated that the same plant defence pathways contribute to both resistance and susceptibility against this necrotrophic pathogen, depending on the timing of their activation. To further understand the molecular mechanisms of petal blight resistance, the role of the phenylpropanoid pathway, identified as a key feature in the transcriptome study above, was investigated in more detail. This pathway produces various metabolites, including phenolic acids, aldehydes, and alcohols, which have numerous physiological functions and also participate in the production of flavonoids and lignin. Resistant C. lutchuensis was shown to rapidly activate the expression of core phenylpropanoid genes after treatment with C. camelliae ascospores. LC-MS-based quantification of phenylpropanoid compounds demonstrated that within the first 6 h of the infection, resistant plants had already accumulated coumaric, ferulic and sinapic acids, while at 24 hpi, concentrations of coumaraldehyde, sinapaldehyde, and caffeyalcohol were significantly increased. Thus, I further hypothesized that the compounds produced by the phenylpropanoid pathway may have fungistatic activity. Indeed, all tested phenylpropanoids inhibited the growth of C. camelliae in agar plates with different efficacy. Moreover, the application of phenylpropanoid compounds, including ferulic and coumaric acids, fully prevented the formation of petal blight lesions on susceptible Camellia ‘Nicky Crisp’ petals. Taken together, it can be concluded that the phenylpropanoid pathway may contribute to the early defence against the petal blight via the rapid production of fungistatic compounds. Ultimately, these compounds could be used to develop natural antifungal sprays to protect susceptible Camellia flowers. The analysis of the C. camelliae secretome using LC-MS/MS detection of proteins showed that the pathogen produces a large number of carbohydrate-active enzymes in liquid culture and plant petals. Injection of these proteins induced necrosis not only in susceptible Camellia petals but also in petals of the resistant species and leaves of non-host Nicotiana benthamiana. It was proposed that these enzymes can contribute to the virulence of the pathogen by inducing cell death and facilitating necrosis propagation. Thus, the early defence responses of resistant Camellia plants may possibly stop the development of C. camelliae before it starts releasing carbohydrate-active enzymes during the necrotrophic step of the infection. Overall, the results of this research further expand our understanding of plant- necrotroph interactions, suggesting that the timing of plant immune responses may be a crucial factor defining the outcome of the necrotrophic infection.Item UV-B priming for disease resistance : the use of UV-B light to reduce susceptibility of lettuce plants to downy mildew disease : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Pathology at Massey University, Manawatū, New Zealand(Massey University, 2019) McLay, EmilyBiotrophic disease is one of the largest causes of decreased yield in horticulture. Integrated Pest Management (IPM) systems are required to control disease in a manner which is effective and sustainable, yet there are still a limited number of new approaches available. Pretreatments of UV-B light (280-320 nm) have been previously observed to reduce plant susceptibility to disease, and may be a potential disease control tool to use as part of an IPM approach. Here, I characterised the capability of UV-B LED technology to reduce susceptibility of a range of lettuce (Lactuca sativa) cultivars to downy mildew disease caused by the obligate biotroph Bremia lactucae. Reductions in disease susceptibility of UV-B-pretreated plants was observed as: delayed disease incidence, reduced visual disease rating and lower B. lactucae conidia count. UV-B-induced reductions to conidia counts were sufficient to reduce the infectivity of the diseased plant. Secondary infections caused by UV-B-pretreated plants exhibited yet further reduced disease severity. UV-B light has been observed to induce a similar gene expression profile to that of disease defence in plants. To determine the mechanism of a UV-B-induced disease defence, similarities between UV-B and disease defence pathways were identified. Analysis of previously published gene expression data revealed similarities in flavonoid-related gene expression between exposure to UV-B light in Arabidopsis thaliana, and resistance to downy mildew (Hyaloperonospora arabidopsidis). The specific role of flavonoids in UV-induced defence was further investigated, with B. lactucae conidia counts of lettuce plants negatively correlated with flavonoid level in a UV-B-dependent manner. LC-MS was used to identify metabolic features which contribute to this correlation, and of these, quercetin 3-O-(6″-O-malonyl)-β-D-glucoside had the strongest negative correlation with B. lactucae conidia count. The direct effect of quercetin 3-O-(6″-O-malonyl)-β-D-glucoside was tested through infiltration into lettuce leaves followed by subsequent downy mildew infection. Decreased B. lactucae conidia count was observed in two lettuce cultivars infiltrated with quercetin 3-O-(6″-O-malonyl)-β-D-glucoside concentrations similar to those induced by a UV-B-treatment. It was concluded that UV-B-pretreatments can decrease disease susceptibility to downy mildew in lettuce, and that this defence is underpinned in part by UV-B-induced phenolics. These findings highlight the opportunity for UV-B morphogenesis to be exploited in the development of next-generation, sustainable disease control tools.Item Insect bioactive capabilities of Epichloë festucae var lolii AR48 infected Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2018) Miller, Taryn AngelaAs the modern world expands and develops, new innovative methodologies for more efficient and environmentally friendly agricultural practices are required. Loss of crops through abiotic (e.g. drought) and biotic (e.g. herbivory) stresses has a major effect on the success of an agricultural industry. For animal production pasture crops are a key aspect of animal husbandry and directly affects yield and health. Symbiotic fungi belonging to the genus Epichloë form associations with cool season forage grasses and have been exploited as a new innovative method for insect pest management. Ryegrass infected with the asexual E. festucae var lolii strain AR48 has insect bioactivity against both the stem boring fly (SBF-Ceradontha australis) and cutworm moth caterpillar (CC -Agrotis ipsilion). The bioactive/s targeting both insects is currently unknown. The aim of this thesis was to identify the gene/s and/or bioactive/s present in AR48 infected ryegrass that have bioactivity against the SBF and/or CC. Two approaches were taken; the known insect bioactive secondary metabolite pathways in Epichloë were investigated in AR48 through bioinformatics and mass spectrometry, and the gene ‘makes caterpillars floppy’ (mcf), encoding an insect toxin like protein, was investigated through reverse genetics and insect bioactivity trials. A new indole diterpene compound (IDT) was identified in AR48 infected plant material and this compound was absent in other Epichloë strains that do not have SBF and CC bioactivity. The same mcf gene allele as that present in the E. typhina mcf model, previously identified as having CC bioactivity, is present and predicted to be functional in AR48. The other Epichloë strains also have mcf genes predicted to be functional, however the mcf allele is different to the bioactive E. typhina mcf model. Overall, this project was able to identify a new IDT compound with potential insect bioactivity as well as identify two Epichloë mcf gene alleles that potentially have differing insect bioactivities.Item Methods of inoculating cypress with Seridium species to screen for resistance and pathogen variability : a thesis presented in partial fulfilment of the requirements for the degree of Master of Applied Science in Plant Health at Massey University, Palmerston North, New Zealand(Massey University, 2003) Tsatsia, Helen TekulaThe cypress species are grown for their timber value, ornamental beauty and shelter. Their existence is threatened by the presence of cypress canker disease caused by fungal pathogens of the genus Seiridium. The long term solution for controlling this disease is to breed for cypress clones that are resistant to cypress canker. Screening for resistance is conducted by artificially inoculating cypress plants with the pathogen's inoculum. This study aimed at developing reliable methods of artificial inoculation that are suitable for New Zealand's climatic conditions. Infection of cypress plants in nature is caused by conidia but mycelial inocula are more commonly used in artificial inoculation. Several methods of inducing sporulation of Seiridium species were investigated Addition of plant substrates was shown to increase sporulation of cultures of Seiridium isolates. Studies comparing the two types of inocula (mycelial plugs and conidial suspensions) showed that mycelium inocula caused a higher percentage of canker lesions than spore inocula. Conidial inocula offer a more consistent pathogenicity. Experiments to determine the effective spore load revealed that the percentage of canker increased with the increase of inoculum load. Pathogenicity varied between species and individual colonies of Seiridium isolates. Infection of cypress in nature is thought to occur through wounds and in this work, wounding was required for infection under both glasshouse and outdoor conditions. Inoculation of the main stem and side branches showed disease symptoms develop more rapidly on side branches than on the main stem. Investigations on in vitro inoculation of tissue cultured plants and excised side shoots showed the possibility of screening cypress ramets under different environmental conditions. Temperature and percentage relative humidity were found to influence the percentage of successful inoculations on cypress plants.Item Molecular analysis of plant innate immunity triggered by secreted effectors from bacterial and fungal pathogens of apple : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Science, Institute of Agriculture and Environment, Massey University, New Zealand(Massey University, 2017) Prokchorchik, MaximIn comparison to animals, plants do not have a dedicated immune system with mobile immune cells to protect themselves. Instead they rely on the innate immunity of each cell. Plant immunity branches into two classical layers: PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity). PTI detects the conserved molecular patterns (PAMPs) associated with pathogens and often can be overcome by pathogens translocating effector molecules into plant cells to inhibit the PTI. ETI, in turn, relies on intracellular receptors that can specifically recognize effectors or their activity and activate a rapid and robust response. The research presented in this thesis is focused on two pathogens of apple plants: the bacterial pathogen Erwinia amylovora (the causal agent of fire blight) and fungal pathogen Venturia inaequalis (the causal agent of apple scab disease). As both bacterial and fungal pathogens deliver effector molecules in order to promote their virulence, ETI engineering is a promising universal strategy to control these pathogens. In Chapter 3, the main aim was to elucidate the requirements and precise mechanism of how an important effector of E. amylovora, AvrRpt2, is recognized by the MR5 disease resistance (R) protein, derived from a hybrid apple Malus x robusta 5. I identified that a fragment of the guardee apple protein RIN4 was required and sufficient and required for MR5 activation. I further identified crucial amino acid residues responsible for this activation. Interestingly, cognate residues in RIN4 guardee homolog from Arabidopsis thaliana are responsible for suppression of the autoactivity of R protein RPS2. These findings led to the proposal of a novel hypothesis for evolutionary guardee adaption to the pool of R proteins present in plants. In Chapter 4, the main focus was to apply newly acquired whole-genome sequencing data of V. inaequalis for identifying the previously mapped AvrRvi8 effector, as well as several novel effectors predicted in silico. The sequences of these effectors were validated by amplification and resequencing of candidate genes from V. inaequalis cDNA. Further functional analysis of the selected gene candidates was performed. In addition, a library of constructs for generating V. inaequalis knock-out strains was prepared for future work. The findings from this thesis expected to be useful for breeders of apple to battle two economically important pathogens devastating the industry. Deployment of the MR5 system in apples should facilitate fire blight resistance in pipfruit and offers the opportunity for further engineering of MR5 to detect other pathogens. Furthermore, the effector library developed for V. inaequalis offers a novel tool for studying both virulence and avirulence mechanisms present in the applescab pathosystem. It is envisaged that further effector research will elucidate authentic targets critical for resistance development in apple.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, MuhammadAs 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.Item The molecular basis of RPS4/RRS-mediated defense activation in Arabidopsis : thesis submitted to Massey University for the degree of Doctor of Philosophy(Massey University, 2017) Newman, Toby EdwardUpon pathogen invasion, each plant cell has the ability to mount an innate immune response. Plants have evolved R genes, which typically encode nucleotide-binding domain and leucine-rich repeat-containing immune receptors (NLRs). The model plant species, Arabidopsis, harbors the paired NLRs, RPS4 and RRS1, the products of which function cooperatively to confer recognition of the Pseudomonas syringae effector, AvrRps4, and the Ralstonia solanacearum effector, PopP2. The exact mechanism underlying RPS4/RRS1-mediated effector recognition remains unclear; therefore, the function of RPS4 and RRS1 was further elucidated. Firstly, by investigating the avirulence activity of natural variants of PopP2 isolated from R. solanacearum strains from across the Republic of Korea, popP2 was demonstrated to be well-conserved and RPS4/RRS1-mediated recognition of PopP2 could tolerate multiple natural polymorphisms in the popP2 sequence. Moreover, a conserved PopP2 EAR motif was identified and characterized; the EAR motif was shown to be required for in planta PopP2 stability and recognition. Secondly, utilizing suppressor of slh1 immunity (sushi) mutants generated in a forward genetic screen on slh1 mutant seeds, insight was gained into the differential requirements for RRS1 auto-activity and effector perception. A leucine-rich repeat (LRR) mutation, L816F, was identified, which affected auto-activity but not effector recognition. Furthermore, a WRKY domain mutation, C1243Y, was identified, which conferred auto-activity with distinct features compared to other known auto-active RRS1 variants. Notably, a TIR mutant harboring a C15Y mutation was identified that impaired RPS4/RRS1 TIR/TIR heterodimer formation and full-length RRS1 function. Finally, an analagous self-association interface (DE) identified in the crystal structure of the TNL, SNC1, was investigated for its role in RPS4 function. It was demonstrated that the DE interface mutations, R116A and M150R, disabled RPS4 TIR domain effector-independent cell death induction and impaired full-length RPS4 signaling.Item Identification and functional analysis of Pseudomonas syringae pv. actinidiae effector-triggered immunity in Nicotiana spp. and Arabidopsis thaliana : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand(Massey University, 2017) Choi, SeraPseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker in commercially important cultivars of kiwifruit (Actinidia delicosa and A. chinensis) worldwide, including New Zealand. Like many gram-negative pathogens, Psa is expected to utilise type III effectors to promote virulence in host plants. In order to better understand Psa effector-triggered immunity and susceptibility, we aimed to investigate multiple molecular characteristics of Psa type III effectors and their recognition mechanisms in model plants, Nicotiana spp. and Arabidopsis thaliana. Nicotiana tabacum and N. benthamiana are widely-used model plants for Agrobacterium-mediated transient expression (agroinfiltration) of effectors for functional characterization. Firstly, we screened multiple characteristics of effectors from two Psa strains, Psa NZ V13 and Psa NZ LV5. The former is a strongly virulent and the latter is a weakly virulent strain in kiwifruit. By using agroinfiltration in Nicotiana spp. to express individual effector proteins, we observed diverse subcellular localisation for Psa effectors. Additionally, we identified multiple Psa effectors that can trigger HR-like cell death (HCD) in both N. tabacum and N. benthamiana. Using virus-induced gene silencing (VIGS), we identified that some Psa effector-triggered HCD requires the immunity regulator SGT1, suggesting that the Psa effector-triggered HCD could be a result of immunity activation. We focused on one Psa NZ V13 effector, HopZ5, which belongs to the YopJ-like acetyltransferase family. HopZ5 triggers hypersensitive response (HR) in Arabidopsis accession, Ct-1. Another Arabidopsis accession, Col-0, does not develop an HR but shows immunity in response to HopZ5. The gene that confers HopZ5-triggered HR in Ct-1 was identified as SOBER1 (SUPPRESSOR OF AVRBST-ELICITED RESISTANCE 1) by using recombinant inbred lines derived from two parental accessions, Ct-1 and Col-0. SOBER1 is a known suppressor of Xanthomonas effector AvrBsT-triggered immunity. Interestingly, AvrBsT also belongs to YopJ family. Uniquely, SOBER1 specifically suppressed HCD triggered by several YopJ-like acetyltransferase effectors in N. benthamiana, including HopZ5 and HopZ3 from Psa. This suggests a common mechanism shared between a subset of YopJ-like acetyltransferase effectors is suppressed by SOBER1. Finally, we identified one Arabidopsis accession, Ga-0, which carries a truncated SOBER1 variant but does not develop an HR upon HopZ5 delivery. Using bulked- segregant analysis of an F2 population derived from a cross between Ct-1 and Ga-0, we mapped the locus conferring HopZ5-recognition in Ct-1 to the upper arm of Chromosome 3.Item Effector-triggered immunity against Pseudomonas syringae pv. actinidiae in nonhost plants : thesis submitted to the Massey University for the degree of Doctor of Philosophy(Massey University, 2017) Jayaraman, JayPseudomonas syringae pv. actinidiae (Psa) is a virulent and highly damaging pathogen causing bacterial canker in all currently commercially important cultivars of kiwifruit (Actinidia spp.). Arabidopsis and Nicotiana spp. plants, however, are nonhosts to Psa. In our course of investigating the various nonhost resistance mechanisms in play against Psa, we identified several sources of resistance against several Psa strains as well as a possible novel virulence mechanism used by Psa and Hyaloperonospora arabidopsidis (Hpa), a b iotrophic pathogen of Arabidopsis. Firstly, we discovered that the highly virulent strain, Psa V13, triggers hypersensitive response (HR) in Arabidopsis in an accession-‐specific manner and that HopZ5PsaV13, a member of the YopJ family of putative acetyltransferases, confers this bacterial avirulence. We also show that the immunity triggered by HopZ5 is independent from HR in the Arabidopsis accession Col-‐0. Through mutagenesis, we show that key amino acid residues predicted for acetyltransferase activity are vital to HopZ5-‐triggered immunity and HR, phenotypes reproduced in Nicotiana spp. Secondly, we identified multiple sources of avirulence for the kiwifruit low-‐ virulence strain, Psa LV5, in Arabidopsis and Nicotiana benthamiana, namely homologs of previously characterized effectors, HopAR1 and HopAB3, respectively. We additionally show that HopAB3 can trigger resistance in cultivated tomato putatively due to a novel recognition by a cultivated tomato homolog (SlPtoB) of the resistance gene Fen. Finally, we identified several nuclear-‐localized effectors from Psa and Hpa that interact with Arabidopsis WRKY transcription factors, different to WRKYs targeted by previously identified AvrRps4 and PopP2. We show that some WRKYs can trigger a cell death response in N. benthamiana when overexpressed and that coexpression of AvrRps4 or PopP2 is able to suppress this cell death response for the WRKYs they interact with. We show that this suppression is associated with suppression of transcriptional activation ability of the WRKY and 7 propose that this mechanism of transcription suppression may be utilized by other Psa and Hpa effectors identified in this study.