The dynamics of molecular components that regulate aphid-plant interactions

Aphids are economically important insect pests, which feed on phloem sap using stylets. Aphids cause significant losses of crop yield, through draining plant resources and vectoring over 275 plant viruses. In plant-pathogen interactions, basal plant defense involving pathogen associated molecular pattern (PAMP) triggered immunity (PTI) and effector triggered immunity (ETI) effectively fends off the majority of plant pathogens. I aimed to discover whether these mechanisms are also involved in the plant response to aphids. I found that elicitors present within aphids can evoke PTI/ETI defense responses. In Arabidopsis thaliana, perception of aphid elicitors requires the Leucine-Rich Repeat Receptor- Like Kinase (LRR-RLK) BAK1, which is required for multiple PTI responses via interaction with other RLKs. I identified two RLKs which may detect aphid elicitors and provide specificity to aphid detection. Successful aphid colonization of plants is thought to involve the suppression of PTI and ETI via effectors, leading to effector-triggered susceptibility (ETS). I investigated a Myzus persicae effector, Mp10, and found that it was required for success on Arabidopsis and could block immune signalling. A plant target for Mp10 was identified via a yeast twohybrid screen. Further investigations suggest that the Mp10 target has previously unknown roles in immune receptor trafficking. Mp10 induces ETI-like responses when expressed in plants, which I found were not dependent upon Mp10 effector action or salicylic acid. A yeast two-hybrid screen of candidate aphid effectors revealed interactions with plant resistance proteins, which may play a role in the aphid-plant interaction. Aphid effector proteins were also found to interact with each other, suggesting a role in the regulation of effector action and delivery into plants. Taken together, the research described in this thesis has elucidated the roles of PTI, ETS and ETI in insect-plant interactions and identified specific plant and aphid proteins that are involved in these

The role of calcium signalling in plant-aphid interactions

Myzus persicae is one of the most successful insects on the planet. It is the world’s most pesticide-resistant insect, feeds on hundreds of plant species and acts as a vector for over 100 viruses. Upon perception of M. persicae feeding, plants activate pattern-triggered immunity (PTI), a pivotal part of which is believed to be calcium signalling. The aim of this thesis is to uncover the role that calcium signalling might be playing in the interaction between M. persicae and one of its hosts: the model plant Arabidopsis. Using a fluorescent calcium sensor (GCAMP3), in vivo imaging of calcium dynamics was performed on leaves infested with M. persicae. There is a rapid and highly localised calcium burst around the feeding site in the epidermal and mesophyll cells, making it as one of the first plant responses to aphid attack. This calcium burst is triggered after perception of the aphid by the defence co-receptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE 1 (BAK1), establishing it as part of PTI. Calcium is released from the extracellular space into the cell by GLUTAMATE-LIKE RECEPTORS 3.3 and 3.6 (GLR3.3 and GLR3.6), in combination with the release of intracellular calcium from the vacuole by TWO-PORE CHANNEL 1 (TPC1). Loss of BAK1, GLR3.3/GLR3.6 or TPC1 significantly attenuates the aphid-induced calcium burst and has an effect on the induction of anti-aphid defence responses. Downstream of the burst, CBL-INTERACTING PROTEIN KINASES 3, 9, 23 and 26 are activated by calcium and together mediate plant resistance to aphid attack. Furthermore, the M. persicae effector Mp10 partially suppresses the feeding site calcium burst, suggesting that the aphid is manipulating this pathway as part of its successful colonisation of the plant. Together, the data presented in this thesis provide evidence for the significant involvement of calcium signalling in the plant response to aphid attack. (Supplementary videos were submitted as separate files which could not be uploaded to the repository. Please contact the author for more information

Identification of genetic modifiers of ACCELERATED CELL DEATH 6 (ACD6) in natural Arabidopsis thaliana accessions

Plants defend themselves against pathogens by activating responses that can also cause unintended collateral damage to the plant itself. Improved understanding of the evolutionary constraints and molecular mechanisms affecting these responses can provide means to minimize the tradeoff between disease-related losses and hyperimmunity-related yield drag in crops. As a model to investigate this problem, I have exploited natural variation at the ACCELERATED CELL DEATH 6 (ACD6) gene, which controls a major trade-off between growth and disease resistance among natural accessions of Arabidopsis thaliana. The hyperactive allele ACD6-Est-1 is known to confer broad-spectrum immunity, but at the same time to also negatively affect growth in many A. thaliana accessions. Here, I first surveyed a large collection of A. thaliana genomes for the presence of Est-like ACD6 alleles. I confirmed that not all accessions with this allele express overt hyperimmunity. I then demonstrated that Est-like ACD6 alleles from accessions that do not show the typical autoimmune phenotype normally associated with this allele could confer hyperimmunity when transformed into a different genetic background, indicating that the attenuation of the Est-like ACD6 phenotype was likely due to extragenic modifiers. I then investigated pathogen responses of several of these accessions more closely. My experiments revealed that reduced growth and immune responses were partially uncoupled in some of these accessions. These findings dovetailed with genetic results suggesting that different accessions contain genetically distinct modifiers of the typical Est-like ACD6 phenotype. Finally, I demonstrated by quantitative trait loci (QTL) mapping that these modifiers are located in different regions of the genome, with one of the modifiers potentially being a gene in cluster of genes encoding nucleotide-binding domain and leucine-rich repeat (NLR) immune receptors. This is an important finding, as ACD6 had previously been linked only to PAMP-triggered immunity (PTI), but not to effector-triggered immunity (ETI), which predominantly relies on NLR immune receptors. My study thus provides new insights into the complex genetic interactions that affect disease resistance and growth

Isolation and functional characterization of Arabidopsis powdery mildew effector proteins

Plants are resistant to the majority of potential pathogenic microbes. Adapted pathogens can however overcome plant defense and induce susceptibility. The molecular processes underlying this adaptation are only partially understood. Obligate biotrophic pathogens, which require a living host for growth and reproduction, establish especially intimate relationships with their plant hosts. A crucial aspect of this lifestyle is the formation of a specialized infection structure termed the haustorium. Haustoria are believed to represent pivotal sites of nutrient uptake and deliver effectors, proteins that manipulate the host cell during infection to promote susceptibility. While the effector arsenal of pathogenic bacteria has been investigated intensively, the repertoires and host targets of fungal effectors are currently underexplored. The work presented here thus aims at characterizing virulence mechanisms employed by the obligate biotrophic Ascomycete Golovinomyces orontii, the causal agent of the powdery mildew disease in Arabidopsis thaliana (hereafter Arabidopsis). To this end, the haustorial transcriptome of G. orontii was obtained by pyrosequencing of a cDNA library generated from isolated haustorial complexes. Transcripts coding for gene products with roles in protein turnover, detoxification of reactive oxygen species and fungal pathogenesis were abundant, while surprisingly transcripts encoding presumptive nutrient transporters were not highly represented in the haustorial cDNA library. A substantial proportion (~38%) of transcripts encoding predicted secreted proteins comprised effector candidates. These candidates were cloned and found to frequently suppress induced plant cell death. A subset of effectors enhanced bacterial virulence and could suppress callose deposition, indicating a role in defense suppression. Transcript profiling of these effectors suggested their sequential delivery during pathogenesis. Furthermore, subcellular localization revealed diverse target compartments in the host. In a complementing approach, a large-scale yeast 2-hybrid (Y2H) assay was performed on the 84 cloned effector candidates and revealed convergence onto 61 potential host targets. These targets were enriched in transcription factors and components involved in development and cellular trafficking. Bimolecular fluorescence complementation assays confirmed the interaction of selected effectors with their host interactors. Finally, the Y2H targets of effectors were used to construct an integrated protein-protein interaction network of Arabidopsis and the three adapted pathogens Pseudomonas syringae (Psy), Hyaloperonospora arabidopsidis (Hpa) and G. orontii. This network revealed pathogen-specific as well as nine common host targets. These common targets are highly connected in the Arabidopsis cellular network. After the development of suitable quantitative methods, the important role of these common targets in the Arabidopsis immune response was validated by screening respective T-DNA insertion lines. In sum, my work supports the hypothesis that pytopathogenic microbes target hubs in the host cellular network to promote susceptibility. The effector targets identified will therefore form the basis of subsequent effector research in G. orontii

UVR8 mediated spatial differences as a prerequisite for UV-B induced inflorescence phototropism

In Arabidopsis hypocotyls, phototropins are the dominant photoreceptors for the positive phototropism response towards unilateral ultraviolet-B (UV-B) radiation. We report a stark contrast of response mechanism with inflorescence stems with a central role for UV RESISTANCE LOCUS 8 (UVR8). The perception of UV-B occurs mainly in the epidermis and cortex with a lesser contribution of the endodermis. Unilateral UV-B exposure does not lead to a spatial difference in UVR8 protein levels but does cause differential UVR8 signal throughout the stem with at the irradiated side 1) increase of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), 2) an associated strong activation of flavonoid biosynthesis genes and flavonoid accumulation, 3) increased GA2oxidase expression, diminished gibberellin1 levels and accumulation of DELLA protein REPRESSOR OF GA1 (RGA) and, 4) increased expression of the auxin transport regulator, PINOID, contributing to local diminished auxin signalling. Our molecular findings are in support of the Blaauw theory (1919), suggesting that differential growth occurs trough unilateral photomorphogenic growth inhibition. Together the data indicate phototropin independent inflorescence phototropism through multiple locally UVR8-regulated hormone pathways

Tree Peony Species Are a Novel Resource for Production of α-Linolenic Acid

Tree peony is known worldwide for its excellent ornamental and medical values, but recent reports that their seeds contain over 40% α-linolenic acid (ALA), an essential fatty acid for humans drew additional interest of biochemists. To understand the key factors that contribute to this rich accumulation of ALA, we carried out a comprehensive study of oil accumulation in developing seeds of nine wild tree peony species. The fatty acid content and composition was highly variable among the nine species; however, we selected a high- (P. rockii) and low-oil (P. lutea) accumulating species for a comparative transcriptome analysis. Similar to other oilseed transcriptomic studies, upregulation of select genes involved in plastidial fatty acid synthesis, and acyl editing, desaturation and triacylglycerol assembly in the endoplasmic reticulum was noted in seeds of P. rockii relative to P. lutea. Also, in association with the ALA content, transcript levels for fatty acid desaturases (SAD, FAD2 and FAD3), which encode for enzymes necessary for polyunsaturated fatty acid synthesis were higher in P. rockii compared to P. lutea. We further showed that the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and α-linolenic acid content, respectively and modulated their final ratio in the seed oil. In conclusion, we identified the key steps that contribute to efficient ALA synthesis and validated the necessary desaturases in P. rockii that are responsible for not only increasing oil content but also modulating 18:2/18:3 ratio in seeds. Together, these results will aid to improve essential fatty acid content in seeds of tree peonies and other crops of agronomic interest

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin