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While activation of resistance (R) proteins has been intensively studied, the mechanisms acting downstream of R protein activation remain elusive. We studied effector-triggered immunity (ETI) conditioned by the potato Ny-1 gene against potato virus Y. Transcriptomic, ultrastructural and biochemical analyses of four consecutive tissue sections in and surrounding the foci of viral infection at three different lesion developmental stages revealed processes that are spatiotemporally regulated. The transcriptional response in the cell death zone and surrounding tissue is dependent on SA. For some of the genes, spatiotemporal regulation is completely lost while for others, the regulation is different in SA-deficient line indicating multiple connections between hormonal signaling modules. The induction of RBOHD NADPH oxidase expression, together with expression of Thioredoxin H gene, occurs specifically in the border region of the lesion during ETI. In plants with silenced RBOHD, ETI is perturbed and virus spread is not arrested at the site of infection. Expression of RBOHD is under the control of SA. On the other hand, RBOHD activity is required for spatial regulation of SA accumulation. We identified an UDP-glucosyltransferase, encoding an enzyme involved in feedback activation of SA biosynthesis, that is derepressed at the border of the lesion in RBOHD silenced plants. Altogether, we revealed a novel aspect of viral ETI, the RBOHD-SA feedback loop required for its spatial regulation. SIGNIFICANCE To get towards sustainable resistance in the field, it is important to understand mechanisms of immune signaling. We here show novel aspects of spatial regulation in effector-triggered immunity (ETI) against potato virus Y. A detailed spatiotemporal analysis in and surrounding the foci of viral infection, on ultrastructural, biochemical and gene expression levels revealed that redox state maintenance is tightly spatiotemporally regulated. We also show the interconnection of salicylic acid and RBOHD through a feedback loop, regulated through UDP-glucosyltransferase, revealing the mechanism of this spatial regulation. The proper function of this loop is essential for successful ETI leading to viral arrest.

V citoloških vzorcih občasno opazimo različne kontaminante, kot so smukec, kristali in nitke bombažne vate. Pri rutinskem pregledovanju citoloških vzorcev urina za malignost smo občasno opazili kontaminacijo preparatov z rastlinskimi celicami. Ugotovili smo, da so bile prisotne samo pri bolnikih, ki so imeli narejen obvod urina po Brickerju. Preparate urina za rutinsko citopatološko preiskavo smo pripravili z membransko filtracijo, jih fiksirali v Delaunayu in jih pobarvali po Papanicolaouu. Pregledali smo jih s svetlobnim mikroskopom. Pripravili smo tudi preparate iz kožnih podlog podjetij Coloplast in ConvaTec, ki jih stomisti uporabljajo za pritrditev vrečke na urostomo. Samo v Coloplastovih kožnih podlogah smo našli rastlinske celice, ki so bile morfološko enake rastlinskim celicam v urinu bolnikov z Brickerjevim mehurjem. Pripravili smo tudi preparate iz različnih snovi, ki so sestavni del kožnih podlog. Rastlinske celice so bile prisotne samo v preparatih guar gumija, ki ga pridobivajo iz semen rastline Cyamopsis tetragonoloba. Rastlinske celice so se po velikosti, obliki in barvi močno razlikovale od epitelijskih črevesnih celic, ki so prisotne v urinu bolnikov z Brickerjevim mehurjem, zato bi jih tudi neizkušen presejalec težko zamenjal z displastičnimi človeškimi celicami. Pomembno pa je, da vse elemente, ki jih najdemo v celičnih vzorcih, prepoznamo in da pri neobičajnih najdbah, kot so rastlinske celice, tudi razložimo, kako je prišlo do kontaminacije vzorca. Cytology samples occasionally reveal different contaminants, such as talc, crystals and cotton wool strings. The routine examination of cytology urine samples to confirm malignancy occasionally showed contamination with plant cell. We have established that they were present only in patients with a Bricker ileal conduit. Urine preparations for the routine cytopathological examination were prepared with membrane filtration, fixed in Delaunay solution and stained according to Papanicolaou. They were examined using the light microscope. We also prepared adhesives preparations made by the companies Coloplast and ConvaTec, which are used by the ostomist to attach the bag on the urostomy. Plant cells were found only in the Coloplast adhesives. Morphologically, they were equal to the plant cells found in the urine of patients with a Bricker ileal conduit. To continue, we also provided preparations from different substances, namely adhesives components. Plant cells were present only in the preparations of guar gum, which is produced from Cyamopsis Tetragonoloba seeds. The size, shape and colour of plant cells differed from those of epithelial intestinal cells. The latter are present in the urine of patients with a Bricker ileal conduit, thus even an inexperienced cytotechnologist would have difficulty confusing them with dysplastic human cells. Nevertheless, it is important to take knowledge of all elements found in cell samples and to provide an explanation for sample contamination in the event of unusual findings, such as plant cells.

V teoretičnem delu diplomske naloge je izpostavljen pomen izvajanja kalitvenih poskusov in katere učne cilje lahko preko njih dosežemo. Potrebno je načrtovati dejavnosti pri katerih učenci samostojno raziskujejo in opazujejo naravo. V empiričnem delu diplomske naloge je bila izvedena krajša raziskava s pomočjo anketnega vprašalnika za učitelje naravoslovja. Preverila sem, če učitelji naravoslovja v osnovni šoli pri poučevanju izvajajo kalitvene poskuse, ter na kakšen način jih izvajajo. Preko eksperimentalnega dela diplomske naloge sem optimizirala izvedbo kalitvenih poskusov, primernih za poučevanje naravoslovja v 6. in 7. razredu osnovne šole in preverila kalivost različnih vrst rastlin. Pri izboru rastlin za kalitvene poskuse sem se osredotočila na vrste, ki so dostopne v semenarnah in trgovinah. V šolskih učbenikih se največkrat prikazuje kalitev in rast fižola ter koruze. Zastavljen cilj je bil najti nekaj dodatnih vrst rastlin, katerih semena so primerna za izvajanje poskusa kalitve pri pouku naravoslovja in jih primerjati z vrstami, ki se že uporabljajo za prikaz kalitve po učnem programu. Izbrala sem 23 vrst rastlin, med njimi 16 dvokaličnic in 7 enokaličnic. Raziskava je pokazala, da je 14 vrst primernih, 3 vrste so manj primerne in 6 vrst rastlin je neprimernih za uporabo pri poskusih kalitve v osnovni šoli. In the theoretical part of my diploma task I focused on the importance of carrying out the germination trials as well as which teaching aims could be attained by such trials. It is necessary to plan the activities by which the pupils self-independently research and monitor nature. In the empirical part of the mentioned task, a short research was carried out by a questionnaire for the teachers of natural sciences. I checked whether the teachers of natural sciences at primary schools carry out the germination trials and the way they do them. By the aid of the experimental part of my task, I optimized the carrying out of the germination trials, which are suitable for teaching natural sciences in the 6th and 7th classes of primary schools and I examined the germination of different vegetable species. I selected the plant for the germination trials according to the species available at the shops and seedman`s shops. Most often, the germination of beans and maize is presented in the text books. My aims was to find some additional vegetable species of which their seeds are suitable for carrying out the germination trials according to the course syllabus. I chose 23 vegetable plants, among them 16 dicotyledonous and 7 monocotyledonous. The research showed that 14 species are suitable, 3 less suitable and 6 unsuitable to use at the germination trials at primary schools.