In silico določitev in mutacijska analiza kandidatnih efektorjev fitopatogene glive Verticillium albo-atrum

Marton, Kristina (author), Javornik, Branka (mentor), Berne, Sabina (co-mentor)

Gliva Verticillium nonalfalfae, prej poimenovana V. albo-atrum, je fitopatogena askomiceta, ki povzroča bolezen prevodnega tkiva različnih rastlin, imenovano verticilijska uvelost. V Sloveniji največ škode povzroča V. nonalfalfae na hmelju (Humulus lupulus). Poleg fitosanitarnih ukrepov je najučinkovitejši pristop preprečevanja bolezni gojenje odpornih sort. Ključnega pomena pri razvoju novih ukrepov je poznavanje dejavnikov patogenosti glive, predvsem njihovih sekretornih proteinov, kot so efektorji, encimi in toksini. V doktorski disertaciji smo se osredotočili na efektorske proteine, ki smo jih želeli določiti z analizo razpoložljivih genomskih in proteomskih podatkov ter rezultatov RNA-Seq eksperimentov ter jih potrditi z mutacijsko analizo. Anotiranim genskim modelom glive V. nonalfafae (9269) smo z bioinformacijskimi orodji najprej določili in silico sekretom (944), ki je obogaten z encimi, ki so vpleteni v razgradnjo celičnih sten, s proteazami, lipazami, kutinazami in oksidoreduktazami, kar sovpada s patogenezo glive V. nonalfalfae kot hemibiotrofom. Nadalje smo identificirali genske modele izražene in planta (766), predvideli kandidatne sekretorne efektorske proteine (263) ter izbrali najboljše kandidate na podlagi razvrščanja po lastnostih že potrjenih glivnih efektorjev. V odpornih in neodpornih rastlinah hmelja po okužbi z V. nonalfalfae smo z RT-qPCR določili ekspresijo najbolj obetavnim (44) kandidatom. Z uporabo ATMT metode smo pripravili delecijske mutante petim izbranim efektorskim genom in umetno okužili neodporne sorte hmelja. Bolezenska znamenja smo ocenili z bolezenskim indeksom ter z izračunanimi vrednostmi rAUDPC. Bioinformacijski postopek za določanje efektorjev se je izkazal za relativno učinkovitega, saj smo z njim potrdili že prej določena efektorja VnaSSP4.2 in VnaUn.279 glive V. nonalfalfae. Delecijski mutant ΔVna1.565 je kazal zakasnelo virulenco, ΔVna8.691 pa povečano virulenco, medtem ko pri ostalih treh delecijskih mutantih nismo zaznali statistično značilnih razlik v primerjavi z divjim tipom glive, kljub njihovi visoki ekspresiji in planta. Njihova potencialna vloga v patogenezi glive je obravnavana v razpravi.

• Language: Slovenian
• Keywords: Verticillium albo-atrum, efektorji, mutacijska analiza, fitopatogena gliva
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Year: 2018
• PID: 20.500.12556/RUL-101957
• COBISS.SI-ID: 926327
• Publication date in RUL: 15.07.2018

Secondary language

• Language: English
• Title: In silico determination and mutational analysis of canidate effectors from phytopathogenic fungus Verticillium albo-atrum

Abstract

Verticillium nonalfalfae, formerly V. albo-atrum, is a phytopathogenic ascomycete, which causes disease of the vascular tissue of various plants named verticillium wilt. In Slovenia, the most damage is done by Verticillium nonalfafae on hops (Humulus lupulus, L.). Apart from phytosanitary measures, the most effective approach for disease control is the cultivation of resistant varieties. The key to the development of new measures is knowledge of the factors of fungal pathogenicity, especially their secretory proteins, such as effectors, enzymes and toxins. In this doctoral study, we aimed to determine V. nonalfalfae candidate effectors, by analysing the available genomic and proteomic data, and the results of RNA-Seq experiments, and to confirm them with mutational analysis. From the annotated genetic models of V. nonalfafae (9269), we initially identified in silico secretome (944) enriched with enzymes involved in the degradation of cell walls, with proteases, lipases, cutinases and oxidoreductases, which corresponds to the hemibiotrophic life style of V. nonalfalfae. We further identified gene models expressed in planta (766), predicted candidate secretory effector proteins (263), and selected the best candidates based on the properties of already confirmed fungal effectors. We determined the expression of the most promising (44) candidates by RT-qPCR in resistant and susceptible varieties of hop after infection with V. nonalfalfae. Using the ATMT method, we prepared five deletion mutants of candidates with the highest expression, and artificially inoculated susceptible varieties of hop. Disease symptoms were assessed with a disease severity index and rAUDPC. The bioinformatic pipeline for determination of effectors proved to be relatively effective, since we determined previously verified V. nonalfalfae effectors VnaSSP4.2 and VnaUn.279. The deletion mutant ΔVna1.565 showed delayed disease symptoms and ΔVna8.691 showed increased virulence, while the remaining three deletion mutants did not show statistically significant differences compared to the wild type fungus, despite their high expression in planta. Their potential role in the pathogenesis of fungi is discussed.

• Keywords: Verticillium albo-atrum, effectors, mutational analysis, phytopathogenic fungus